Mullis was born in Lenoir, North Carolina, near the Blue Ridge Mountains, on December 28, 1944. His family had a background in farming in this rural area. As a child, Mullis recalls, he was interested in observing organisms in the countryside. He grew up in Columbia, South Carolina, where he attended Dreher High School. He has described his early interest in chemistry, learning how to chemically synthesize and build solid state fuel propulsion rockets as a high school student during the 1950s.
Mullis earned a B.S. in chemistry from the Georgia Institute of Technology in Atlanta in 1966, during which time he got married and started a business. He then received a Ph.D. in biochemistry from the University of California, Berkeley in 1973; his research done in J. B. Neilands' laboratory focused on synthesis and structure of bacterial iron transporter molecules. Following his graduation, Mullis completed postdoctoral fellowships in pediatric cardiology at the University of Kansas Medical Center (1973-1977) and pharmaceutical chemistry at the University of California, San Francisco (1977-1979).