Congenital syphilis (CS) is caused by Treponema pallidum infection in utero. There is a need to develop new diagnostic tools for CS: the diagnostic value of PCR is difficult to assess. The aim of this study was to describe the clinical and laboratory characteristics of mothers and infants with CS as diagnosed by PCR tests on various maternal and neonatal samples. Patients and methods: The study included all infants epidemiologically linked to a mother diagnosed with syphilis whose samples were referred to the Syphilis Reference Center, and for whom at least one positive PCR result was obtained. Results: Twenty-two mother-infant pairs (8.3%) with assays performed on samples from one to four different anatomical sites were included between February 2011 and April 2018. Seven mothers (31.8%) were born abroad, fifteen (68.2%) had psychological and/or social problems, eight (36.4%) had not been screened for syphilis prior to delivery, and eleven (50%) were referred from French overseas departments or territories, or from the Paris region. Six infants (27.3%) were stillborn and six were born preterm, while fifteen infants (68.2%) presented clinical features of congenital syphilis. All infants born preterm were symptomatic. Infant VDRL/RPR titers were no greater than four times those in the mother’s serum, except in two cases. Discussion: lack of antenatal care, social disadvantage, and psychological issues were common. There is a need for enhanced surveillance both in the French overseas departments and territories and in the Paris region. A larger study is required to assess the sensitivity and specificity of PCR. The optimal site for sampling has yet to be established. We recommend collecting as many samples as possible to avoid underdiagnosis of CS.