Introduction. Syphilis is a disease produced by Treponema pallidum subspecies pallidum, which affects approximately 12 million people worldwide every year. Of these, more than 2 million are pregnant women whose babies end up having congenital syphilis, the worst form of this infection.Objective. To detect the presence of T. pallidum subspecies pallidum in clinical samples in order to diagnose congenital syphilis by means of nested PCR, and to determine its concordance with serological testing.Materials and methods. Three target genes (polA, 16S ADNr y TpN47) were amplified by conventional and nested PCR. The results from the amplification of the TpN47 and polA genes were confirmed by sequencing. The serological tests used were VDRL (Venereal Disease Research Laboratory), RPR (Rapid Plasma Reagin) y TPPA (Treponema pallidum Particle Agglutination Assay).Results. The sensitivity for the conventional PCR was 52 pg and 0.52 pg for the nested PCR. The specificity of primers TpN47 and polA was 100 %; the results of the sequencing showed a 97 % identity with T. pallidum. There was concordance between the serology and the nested PCR in 70% of the samples.Conclusion. The TpN47 gene was the best molecular target for the identification of T. pallidum. The nested PCR is a promising molecular tool for the diagnosis of congenital syphilis.