The aim of this study was to determine if a reservoir of sub-clinical LGV infection exists in men who have sex with men (MSM), as this finding might account for the recent rise in lymphogranuloma venereum (LGV) Chlamydia trachomatis infections among MSM in Canada. MSM without proctitis were enrolled between January and August 2006 in a cross-sectional study. Rectal, urine, serology and pharyngeal specimens were tested for specific C. trachomatis serovars. The median age of the 253 participants was 43 years; 53% were HIV+. We found no active cases of LGV infection; but 20 (8%) participants had positive serology. Thirteen participants (5%) had non-LGV C. trachomatis infections. Unprotected anopenetrative intercourse, rectal enema and drug use were associated with non-LGV C. trachomatis infection. Sub-clinical rectal non-LGV C. trachomatis infection was relatively common but LGV was not identified in our sample. Further studies of screening for non-LGV chlamydia infection in MSM are needed.