In India, many state reference centres for sexually transmitted infections perform only a single screening assay for syphilis diagnosis. In this study, Treponema pallidum haemagglutination (TPHA) was performed on 1115 Venereal Disease Research Laboratory (VDRL)/rapid plasma regain (RPR) non-reactive and 107 reactive sera out of 10,489 tested by VDRL/RPR according to the National AIDS Control Organisation syphilis testing protocol. A total of 47 Specimens reactive in TPHA and non-reactive with VDRL test were subjected to fluorescent treponemal antibody absorption and enzyme-immunoassay. Seroprevalence considering both VDRL and TPHA positivity was highest (4.4%) in sexually transmitted diseases clinic attendees than in other subject groups. Positivity by two treponemal tests in 24 (2.2%) cases non-reactive by VDRL/RPR was representative of the fully treated patients or latent or late syphilis cases. The findings highlight that a suitable treponemal confirmatory test should be performed in all the diagnostic laboratories.