Objectives: To assess the prevalence and associated risk factors of syphilis among antenatal clinic attendees by a multi-center cross-sectional study in Karachi, Pakistan. Methods: We administered a structured questionnaire and obtained a blood sample for syphilis serology (rapid plasma reagin test with Treponema pallidum hemagglutination assay confirmation) from all women giving informed consent over six weeks in 2007. Prevalence was calculated at 95% confidence intervals. Multivariate analysis was adapted to assess risk factors. Results: There were seven (0.9%) confirmed cases of syphilis (95%CI: 0.4, 1.8) in a sample size of 800 women recruited from three urban sites (~1% refusal rate). Women who lived in an area where male drug use is prevalent had 1.5% higher prevalence rates than women from the other two sites 0.5%. Conclusions: We documented higher-than-expected syphilis seroprevalence rates in a low risk population of antenatal clinic attendees in Pakistan. Bridge populations for syphilis may include drug users, who are usually married, and Hijras or their clients. In accordance with our results, the national policy for syphilis control in Pakistan should be modified to include universal syphilis screening in antenatal clinics with subsequent partner notification.