Background Congenital syphilis is recognized as a substantial public health problem in Sub-Saharan Africa. The aim of this study was to determine the prevalence of syphilis among antenatal care attendees in Botswana and to contribute to knowledge about the challenges facing the syphilis-screening programme. Methods In a cross-sectional study, 703 antenatal care attendees at 13 health facilities in Gaborone, Botswana were interviewed and examined. Venous blood samples were collected for the identification of syphilis infection. The antenatal records were used to obtain information on any screening, diagnosis and treatment of syphilis that had been done earlier in the current pregnancy. Results Active syphilis was found in 32 (5%) of the attendees. Among 546 women coming for a repeat antenatal care visit, 71 (13%) had not been screened for syphilis. Uptake late in pregnancy, delayed treatment and a high rate of seroconversion after testing were other identified obstacles to the effective prevention of congenital syphilis. Conclusion Syphilis prevalence among pregnant women in Botswana remains high, and there is still much to be gained by improving the effectiveness of the syphilis screening and treatment programme. Earlier antenatal care attendance, rapid on-site testing, improved partner treatment and a repeat test late in pregnancy to manage incident cases are important goals for patients, health care workers and health authorities.