Kilrain was born in Augusta, Georgia, as Susan Leigh Still to Dr. Joseph Still, M.D. and his wife, Jean Ann Batho; she has nine siblings. Her father was a prominent burn surgeon who founded and directed the Joseph M. Still Burn Center in Augusta, Georgia. Kilrain graduated from the Walnut Hill School, Natick, Massachusetts, in 1979. She graduated from Embry-Riddle Aeronautical University in 1982 with a bachelor's degree in aeronautical engineering and received her Master of Science degree in aerospace engineering from Georgia Institute of Technology in 1985.

She is married to Vice Admiral Colin J. Kilrain, who previously served with North Atlantic Treaty Organization Special Operations Commander Headquartered in Belgium. Kilrain and her husband have four children, and reside in Virginia Beach, Virginia. She is active as a motivational speaker, presenting mostly to schools and universities. Her main message is "Live Your Dream" - anyone can become an astronaut.

After graduation, Kilrain worked as a wind tunnel project officer for Lockheed Corporation in Marietta, Georgia, and earned her graduate degree. She was commissioned into the U.S. Navy in 1985 and designated a Naval Aviator in 1987. Kilrain was selected to be a flight instructor in the TA-4J Skyhawk, and later flew EA-6A Electric Intruders for Tactical Electronic Warfare Squadron 33 (VAQ-33) in Key West, Florida. After completing U.S. Naval Test Pilot School at NAS Patuxent River, Maryland, she reported to Fighter Squadron 101 (VF-101) in Virginia Beach, Virginia, for F-14 Tomcat training. Kilrain has logged over 3,000 flight hours in more than 30 different aircraft.

Kilrain reported to the Johnson Space Center as an Astronaut Candidate in March 1995. Following a year of training, she worked on technical issues for the Vehicle Systems and Operations Branch of the Astronaut Office. She also served as spacecraft communicator (CAPCOM) in mission control during launch and entry for numerous missions. A veteran of two space flights, she logged nearly 472 hours in space. She flew as pilot on STS-83 (April 4 to April 8, 1997) and STS-94 (July 1 to July 17, 1997). She was most recently the Legislative Specialist for Shuttle, for the Office of Legislative Affairs at NASA Headquarters, Washington D.C. Kilrain retired from the Astronaut Office in December 2002 and from the U.S. Navy in 2005.