Aims of the study were to evaluate Human Papillomavirus (HPV) and type-specific prevalence in four anatomical sites in HIV infected men who have sex with men (MSM) compared with HIV uninfected MSM. Participants were recruited among the attendees of Infectious Diseases Clinics in Central Italy. A trained medical practitioner collected by interview sociodemographic data and information on medical history, sexual behavior, and drug use. Swabs from anal canal, oral cavity, urethral mucosa, and coronal sulcus were tested for HPV DNA and genotyping. Ninety MSM were enrolled, 45 subjects within each group. Overall, 48.9% MSM were HPV positive and prevalence was higher in HIV infected men (60.0% vs 37.8%, P = 0.035). HPV at multiple anatomic sites occurred in 59.1% MSM, with 34.1% and 22.7% at two and three sites, respectively. Prevalence of anal, coronal sulcus, oral, and urethral HPV was 96.3%, 37%, 21.6%, and 18.5% in HIV infected MSM, and 70.6%, 70.6%, 29.4%, and 23.5% among HIV uninfected. A similar proportion of HIV infected and uninfected MSM (59.2% and 58.8%) carried at least one high-risk genotype. Prevalence of types covered by nonavalent vaccine was 77.8% in HIV infected compared with 82.3% in HIV uninfected MSM. HPV 58 and 16 were mostly detected in HIV positive (43.7% and 31.2%) and negative MSM (50.0% and 40.0%). HPV detection rate underlined the high vulnerability of MSM to acquire multisite infections, characterized by various genotype combinations. Since nonavalent vaccine could have prevented 80% of HPV infections, study findings support the implementation of vaccination programs among MSM.