Background: Chlamydia trachomatis is a common bacterial sexually transmitted infection in men who have sex with men (MSM), although little is known about its distribution in Australian MSM communities. Methods: From 2004 to 2008, 612 consecutive C. trachomatis positive anal swab and urine samples were collected for genotyping and quantification from MSM attending 2 sexual health centers (Melbourne and Sydney). Results: The most common serovars detected were D (35.2%), G (32.7%), and J (17.7%), although these distributions changed significantly by year and city. C. trachomatis infections (2.8%) involved more than 1 serovar and only 1 lymphogranuloma venereum isolate was detected. The majority of serovar strains showed an identical omp1 genotype, with only 7.5% showing genotypic variability. Serovar G infections were not associated with overseas sexual activity; whilst individuals with serovar J were less likely to have had a prior C. trachomatis infection, and with serovar E were those who had prior C. trachomatis infection. Symptoms were present in 68% of urethral infections and 28% anal infections, and were associated with gonorrheal coinfection (13.8%), prior C. trachomatis infection (20.6%) and increasing age. A higher C. trachomatis load was identified in anal samples versus urine (1.48 × 10 genome copies/anal swab; 3.72 × 10 copies/mL urine) and no association was made to concentration including the presence of symptoms and prior C. trachomatis infection. Conclusions: This is the largest study of C. trachomatis serovars in MSM: it is the first to report C. trachomatis rectal loads, and provides an overview on C. trachomatis serovars and genotypic variants that circulate in Australian MSM communities.