Introduction The World Health Organization (WHO) recommends universal syphilis testing in pregnancy as part of basic maternal and child health services. Coupled with HIV testing, antenatal syphilis testing is fundamental for the regional initiative on elimination of mother-to-child transmission of HIV and congenital syphilis (EMTCT) in the Americas and globally. We conducted this survey of laboratory practices around syphilis testing to characterise syphilis testing in the Pan American Health Organisation (PAHO) member countries. Methods A structured survey assessing syphilis laboratory testing practices in the 35 PAHO member states was administered electronically between March and August 2014. Leaders of national reference labs, large regional laboratories and a sample of local (e.g., large maternal hospitals, district hospitals) and private laboratories that conducted syphilis testing were recruited to participate. The survey collected data on laboratories tests used, testing algorithms applied in different clinical settings, testing volume and turnaround time, quality assurance strategies, and results reporting to national surveillance. Results Data were obtained from 30 (86%) PAHO member states, including 36 national or regional reference laboratories and 33 lower level laboratories, primarily (94%) publicly funded. Of 69 laboratories reporting results, 41% used rapid syphilis tests (RSTs), of which 36% were lower level laboratories. Sixty-eight percent of reporting laboratories (83% of national/regional) participated in external quality control, and 36% reported surveillance data. Of the 69 laboratories, 49 (71%) reported using a national algorithm for syphilis testing in pregnancy, of which 5 involved RSTs. Of 54 (78%) laboratories that reported processing samples from antenatal clinics, approximately half experienced stock outs of at least one essential commodity during the previous 12 months. Conclusions Updating laboratory algorithms along with improving testing standards and quality assurance, supply distribution, and surveillance reporting could better advance EMTCT of syphilis and improve syphilis testing in various clinical settings in the Americas Region. Disclosure of interest No grants or outside funding were received in the development of this study.