Objective: To first test the hypothesis that the presence of viral nucleic acid in amniotic fluid (AF) is associated with an abnormal pregnancy outcome, and second, to determine if the overall rate of polymerase chain reaction (PCR) positivity and the distribution of virus types vary geographically. Study Design: Cytomegalovirus (CMV), parvovirus B19, adenovirus, enterovirus, herpes simplex virus, Epstein-Barr virus, and respiratory syncytial virus nucleic acids were sought in 423 AF samples obtained for clinical indications: 284 from the East Coast (EC) and 139 from the Midwest (MW). Results: Gestational age at sampling was 19.1 weeks for EC and 20.1 weeks for MW. 13.5% of karyotypically normal singleton pregnancies (57/423) had a positive AF PCR. 11% of AF PCR from the EC while 18% of AF PCR from the MW were positive (p = 0.06). The most commonly detected viruses were adenovirus (77%), enterovirus (12%), and CMV and parvovirus B19 (5% each). Twenty-four percent of sonographically abnormal pregnancies (33/136) had a positive AF PCR compared to only 8.4% (24/287) of normal pregnancies (p < 0.001). Conclusion: A positive AF PCR is associated with an increased rate of fetal structural malformations, intrauterine growth restriction, hydrops and other fetal abnormalities. There were no significant geographic differences in the incidence of AF viral PCR positivity.