Objective: To determine contribution of reinfection with new strains of cytomegalovirus in cytomegalovirus seromimmune women to incidence of congenital cytomegalovirus infection. Study Design: In 7848 women studied prospectively for congenital cytomegalovirus infection from a population with near universal cytomegalovirus seroimmunity, sera from 40 mothers of congenitally infected infants and 109 mothers of uninfected newborns were analyzed for strain-specific anticytomegalovirus antibodies. Results: All women were cytomegalovirus seroimmune at first prenatal visit. Reactivity for 2 cytomegalovirus strains was found in 14 of 40 study mothers and in 17 of 109 control mothers at first prenatal visit (P = .009). Seven of 40 (17.5%) study women and 5 of 109 (4.6%) controls (P = .002) acquired antibodies reactive with new cytomegalovirus strains during pregnancy. Evidence of infection with more than 1 strain of cytomegalovirus before or during current pregnancy occurred in 21 of 40 study mothers and 22 of 109 controls (P < .0001). Conclusion: Maternal reinfection by new strains of cytomegalovirus is a major source of congenital infection in this population. © 2010 Mosby, Inc. All rights reserved.