Lewellys F. Barker was born in Baltimore, Maryland in 1933. His grandfather, also named Lewellys F. Barker (1867–1943) was an esteemed physician and professor at Johns Hopkins University. He attended Princeton University for his undergraduate studies and then Johns Hopkins University for medical school, graduating in 1959. He completed his internal medicine residency at Bellevue Hospital in New York City.

Dr. Barker first came to the NIH in 1962 through the Public Health Service. He was assigned to the Laboratory of Virology and Rickettsiology in the Division of Biologics Standards (DBS). He worked first with Dr. Joseph Smadel on rickettsial vaccines, and then with Dr. Harry M. Meyer, Jr. in the DBS Laboratory of Viral Immunology.