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Background Chlamydia trachomatis (CT) is one of the most commonly diagnosed asymptomatic bacterial cause among sexually transmitted infection (STI) worldwide. We assessed the prevalence of CT in random STI patients to evaluate the presence of CT as either a single infection or in co-infection with other STI. Methods A total of 422 urogenital samples were collected from patients who attended the Diagnostic Centre in Saratov Region to be tested for specific DNA of CT and other STIs (Neisseria gonorrhoeae/Trichomonas vaginalis/Mycoplasma hominis/Mycoplasma genitalium/Human papillomavirus (HPV 16/18)/Cytomegalovirus/Herpes simplex virus (HSV 1/2)/Candida albicans/Gardnerella vaginalis/Ureaplasma species. Each clinical sample was carefully screened with the use of commercial kits, either as conventional PCR targeting CT plasmid, or real-time multiplex set (Vector-Best, Russia) validated further by additional confirmatory PCR for the CT-positive samples. Results CT was detected in 17/194 (4,02%) patients who were screened only for the presence of CT DNA (194/422, 45,97%). However, when all 422 patients were systematically screened for CT, along with all other STIs, CT infections were significantly higher (about 4-fold larger) in the patients with other STIs (70/422, 16.58%). Moreover, 56/70 (80%) CT DNA samples were successfully genotyped as CT genovars: E (50%), G (21,42%), D (17,85%), J (5,35%) and K (3,57%). Conclusion Chlamydial asymptomatic infection cases can be frequently masked by clinical symptoms of other STIs. Diagnostic testing for multiple STIs should provide a broader diagnostic coverage for asymptomatic CT patients in order to improve significantly CT early detection, prevention of transmission, and treatment strategies. Disclosure No significant relationships.