Jana Novotná turned professional in February 1987. In the early years of her career, she was known primarily for her success as a doubles player. In the early 1990s, Novotná began to have success in singles once four-time Grand Slam singles champion Hana Mandlíková became her coach. Mandlíková would coach her for nine years. Previously she had been coached by Mike Estep.
At the 1990 French Open, Novotná, seeded 11th, achieved her best results in Grand Slam singles play up until that point. Having reached the round of 16, she faced Argentinian Gabriela Sabatini (seeded 4th). In their four previous meetings, Sabatini got the best of Novotná in three of those matches, including two straight set wins. This time proved to be different, as Novotná turned the tables against Sabatini. Although Novotná had disposed of Sabatini, she would have to face yet another difficult opponent in the quarterfinals, Katerina Maleeva (seeded 8th) from Bulgaria. In their two previous meetings Novotná had lost both times, and after Maleeva won the opening set, it appeared Novotná was about to lose a third consecutive time to Maleeva. However, Novotná came back to defeat Katerina Maleeva. Despite her success, Novotná's toughest test would be against top seeded Steffi Graf of Germany in the semifinals. When Novotná faced Graf three years before at the 1987 French Open, Graf won in straight sets. Graf again defeated Novotná without dropping a set. She qualified for the first time for the season-ending Virginia Slims Championships in which she was beaten in the first round by Sabatini. She finished the year ranked No. 13.
Novotná enjoyed an excellent start to the 1991 season at the Australian Open, where she was seeded 10th and beat Zina Garrison-Jackson 7–6, 6–4 in the round of 16 to advance to the quarterfinal. The path to the final became considerably more difficult, as Novotná had to contend with top seeded Steffi Graf in the quarterfinal encounter. In their ten previous meetings, Novotná had lost each time against Graf.
But this time Novotná pulled the upset of her life by defeating Graf, the reigning champion of the last three years, 5–7, 6–4, 8–6. Now just one win away from her first ever Grand Slam final, Novotná would have to stop Arantxa Sánchez Vicario in the semifinals to get there. Novotná defeated Sánchez Vicario, setting up a showdown against Monica Seles in the final. Seles eventually won in three sets. At the end of the year Novotná was ranked No. 7.
Two years later, at Wimbledon, Novotná's game hit full stride, as she played some of her finest tennis ever. But for Novotná (seeded 8th) to capture the title, her path would have to go through Sabatini (seeded 4th), Martina Navratilova (seeded 2nd) and top-seeded Steffi Graf. Going into her quarterfinal against Sabatini, Novotná had lost six consecutive matches against the Argentine. This time, Novotná took Sabatini apart in straight sets, prevailing 6–4, 6–3. After Novotná got rid of Sabatini, she then set her sights on a semifinal clash against Martina Navartilova, who had won each of their previous five matches. However, Novotná defeated Navratilova, setting up the Championship match against Graf. After losing a tight first set, Novotná took the second set and then had a game-point serving at 4–1 in the third set. With victory seemingly in her grasp, she lost her nerve, double-faulted, and allowed Graf to climb back into the match. Graf took the next five games and the title. During the prize presentation ceremony, a distraught Novotná burst into tears and cried on Katharine, Duchess of Kent's shoulder. The Duchess comforted her. Novotná achieved a year-end ranking of No. 6.
She began the 1994 season by reaching the quarterfinals of the Australian Open. In her quarterfinal match, she played Gabriela Sabatini, in what turned out to be their last head-to-head encounter. In their previous meeting, on the grasscourts at the 1993 Wimbledon Championships, Novotná beat Sabatini in a straight-set quarterfinal victory. This time, on the Australian hardcourts, Sabatini defeated Novotná in straight sets. At the French Open, Novotná was beaten in the first round by Anna Smashnova in straight sets. At Wimbledon, Novotná reached the quarterfinals where she again faced Martina Navratilova in a rematch from the previous year. Novotná lost in three sets. At the season's final Grand Slam, the US Open, the 7th seeded Novotná worked her way to the semi-finals where she played top-seeded Steffi Graf. Dating back to the 1992 French Open, Novotná had lost nine consecutive matches against Graf. This trend would continue as Graf defeated Novotná in straight sets.
It took four years for Novotná to reach another Wimbledon final. In 1997, she faced top-seeded Martina Hingis, and lost in three sets. But to get back to the final, Novotná had to get past Mary Joe Fernandez in the round of 16. She outlasted Fernandez then defeated Yayuk Basuki of Indonesia in the quarterfinals in straight sets. Now back in the semifinals of Wimbledon, her next opponent would be Arantxa Sánchez Vicario. She had prevailed against Novotná in seven of their nine previous contests, but the majority of those matches were played on clay and hard-courts, at which Sánchez Vicario excelled.
On grass, Novotná had a decided advantage, as she defeated Sánchez Vicario in straight sets. In the final against Hingis, she started out fast, taking the opening set 6–2. But Hingis found her stroke and won the second set 6–3 to even the match at one set apiece. The final set proved to be a mirror image of the second set, as Hingis prevailed 6–3, handing Novotná her second loss in a Wimbledon Championship match.
However, Novotná won the Tour Championships after a victory in the final over Mary Pierce and finished the year ranked a career-high world No. 2 in singles. In addition to winning the Tour Championship, Novotná captured three more WTA singles titles for the year.
1998: Wimbledon victory
Her moment of Wimbledon success finally arrived in 1998. After defeating Venus Williams in a close quarterfinal, Novotná avenged the previous year's loss by ousting Martina Hingis in the semifinal in straight sets. She won the singles title by defeating veteran Nathalie Tauziat in the final in two sets. She became the oldest first-time Grand Slam singles winner in the Open Era at age 29 years and nine months. This record would be eclipsed by Francesca Schiavone in 2010 when she won the French Open at 29 years and eleven months and again by Flavia Pennetta when she won the US Open in 2015 at 33 years and 6 months.
Novotná won 12 Grand Slam women's doubles titles (four at Wimbledon, three at the French Open, three at the US Open, and two at the Australian Open) and four mixed-doubles titles (two at the Australian Open, one at Wimbledon, and one at the US Open).
Novotná was a member of the Czechoslovakian team that won the Fed Cup in 1988. At the Olympic Games, Novotná was a women's doubles silver medalist in 1988 and 1996 and a singles bronze medalist in 1996.
She won titles on all four surfaces and crossed the $10 million mark in career prize money in 1998, the fifth player to reach the milestone. She won more than 500 career singles matches, the 15th woman in the Open Era to accomplish the feat.
Novotná was named the 1998 WTA Tour Doubles Team of the Year with Martina Hingis, the 1997 International Tennis Federation Doubles Team of the Year with Lindsay Davenport, the 1996 WTA Tour Doubles Team of the Year with Arantxa Sánchez Vicario, with Gigi Fernández in 1991 and in 1989 and 1990 with Helena Suková.