Anal cancer is a rare cancer but its incidence is increasing. Human papillomavirus (HPV) infection seems to be associated with the occurrence of most cases. The genotype-specific prevalence of HPV in anal cancer was estimated to assess the potential benefit of HPV vaccination in France. Anal cancer histological specimens were retrospectively recruited in 2008 from 16 French centres and centrally tested for HPV genotyping using the INNO-LiPA assay allowing the detection of 28 genotypes. Results were analyzed according to age, gender, HIV status when available and histological diagnosis. A total of 366 anal cancer cases were analyzed among which 62% were females. Mean age at diagnosis was 54.8 years in males and 66.4 years in females (p < 0.001). HPV was found in 96.7% of cases, 72% being infected by a single HPV type. Presence of at least one high-risk genotype was observed in 91% of cases (96% in females and 83% in males; p < 0.001). HPV16 was by far the most prevalent genotype (75%), followed by HPV18, HPV52, HPV33, and HPV51 (4-6%). HPV16/18 alone or in association were found in 78% of all cases. HIV-positive cases had a higher proportion of multiple HPV infection than HIV-negative cases and a slightly different HPV type distribution with an under-representation of HPV16 and an over-representation of other types. Our results indicate that anal cancer rarely occurs in the absence of HPV and emphasize the predominant role of HPV16. The potential benefit of HPV vaccine on the occurrence of anal cancer should be further evaluated.