BackgroundChlamydia trachomatis (CT) infection is one of the most pervasive sexually transmitted infections and has high prevalence in urogenital and extra-urogenital sites among men who have sex with men (MSM). This study investigated anatomical site-specific prevalence and genotypes of CT among MSM recruited from three geographic areas in China.MethodsWe collected urine specimens and anorectal, pharyngeal swab specimens from 379 MSM. CT infection was identified using polymerase chain reaction and CT genotyping was determined by sequences of the ompA gene.ResultsThe results indicated that the overall prevalence of CT infection was 18.2% (95% confidence intervals [CIs], 13.9–22.5%) and significantly different between the cities (p = 0.048). The infection was most common at the anorectal site (15.6, 95%CIs 11.6–19.5%) followed by urethral (3.2, 95%CIs 1.4–5.0%) and oropharyngeal sites (1.6, 95%CIs 0.3–2.9%). Genotypes D and G were the most common CT strains in this population but genotype D was significantly predominated in Nanjing while genotype G was in Wuhan. No genotype related to lymphogranuloma venereum was found. CT infection was significantly related to the infection of Neisseria gonorrhoeae (adjusted odds ratio [aOR] 14.27, 95%CIs 6.02–33.83, p < 0.001) and age. Men older than 40 years old were less likely to have a CT infection as compared to men under 30 years old (aOR 0.37, 95% CIs 0.15–0.93, p = 0.03).ConclusionThe high CT infection prevalence, particularly in the anorectal site, among MSM suggests the necessity to development an integrated CT screening and treatment program specifically focusing on this high-risk population. Surveillance of CT infections should be improved by including both infection and genotype based surveys into the current surveillance programs in China.