An increase in the circulation of HIV-1 non-B subtypes has been observed in recent years in Western European countries. Due to the lack of data on the circulation of HIV-1 non-B subtypes among European HIV-1-infected men who have sex with men (MSM), a biomolecular study was conducted in Rome, Italy. HIV-1 partial pol gene sequences from 111 MSM individuals (76 drug naive and 35 drug experienced) were collected during the years 2004-2006. All these sequences were analyzed using the REGA HIV-1 Subtyping Tool, and aligned using CLUSTAL X followed by manual editing using the Bioedit software. A BLAST search for non-B subtype sequences was also performed. Twenty-six (23.4%) MSM were not Italians. Eight individuals (7.2%) were diagnosed as HIV infected before 1991, 20 (18.0%) between 1991 and 1999, and 83 (74.8%) from 2000 to 2006. Fifteen (15/111, 13.5%) individuals were infected with the non-B subtype. The percentage of infection with HIV-1 non-B subtypes was 8.2% (7/85) among Italian MSM and 30.8% (8/26) among the non-Italians (OR = 4.95 95% IC: 1.40-17.87). Individuals infected with the non-B subtype were significantly younger than those infected with the HIV-1 B subtype (28 years vs. 34 years, p = 0.003). The CRFs were more prevalent (8.1%) than pure subtypes (5.4%), which were distributed as follows: subtype C (2.6%), subtype A1 (1.7%), and subtype F1 (0.9%). Major mutations conferring resistance to antiretroviral drugs (ARV) were not found among HIV-1 non-B subtype drug-naive patients but were found in two ARV-experienced individuals. The data show that viral diversity is likely increasing in a population group that had been previously characterized by the circulation of HIV-1 subtype B.