Mary-Dell Chilton was born in Indianapolis, Indiana, on February 2, 1939. After receiving a B.S. in chemistry from the University of Illinois, Urbana, Mary-Dell Chilton continued to study at the University of Illinois, completing her Ph.D. in Chemistry in 1967 with a dissertation entitled She then went on to complete post-doctoral fellowships at the University of Washington, Seattle, until 1971, when she received a faculty position at the same university. Chilton remained at the University of Washington until 1979 when she accepted a faculty position at Washington University in St. Louis. Chilton's research teams at the universities first demonstrated that the genes responsible for causing disease could be removed from the bacterium without adversely affecting its ability to insert its own DNA into a plant cell. Chilton and her collaborators at Washington University were also the first to produce transgenic plants.

In 1983, Mary-Dell Chilton left Washington University and accepted a position in the private sector at CIBA-Geigy Corporation, which later became Syngenta Biotechnology, Inc. (SBI) in Research Triangle Park, North Carolina. Her tenure there spanned both research and administrative roles, including vice president of agricultural biotechnology, distinguished science fellow, and principal scientist. In 2002, Chilton's contributions to the development of plant biotechnology earned her the Benjamin Franklin Medal in Life Sciences for 2002.

Chilton's current research (as of August 2006) is directed at improving the technology for introducing new genes into plants.

Mary-Dell Chilton was married to Dr. Scott Chilton, who was a professor of botany in the College of Agriculture and Life Sciences at North Carolina State University from 1983 to 2004. In 2004, a few weeks before Scott Chilton's untimely death, Mary-Dell Chilton and her husband completed plans to create an endowment supporting the collections of the NCSU Libraries.