Objectives: Anal cancer is a rare disease. However, its incidence is increasing in some population groups. Infection caused by Human Papillomavirus (HPV) is strongly associated with the risk of anal cancer, whose variability depends on samples, histology, and HPV detection methods. The aim of the study was to assess prevalence and distribution of HPV genotypes in patients diagnosed with anal carcinoma. Methods: An observational, retrospective study was carried out in a tertiary care hospital in North Sardinia, Italy. Specimens of anal cancer diagnosed from 2002–2018 were selected. Demographic, epidemiological, and clinical variables were collected to assess their relationship with the occurrence of anal cancer. Results: The overall HPV positivity was 70.0% (21/30), with HPV-16 being the predominant genotype (~85%). The highest prevalence of anal cancer was in patients aged ≥55 years. HPV positivity was higher in women (p-value > 0.05) and in moderately differentiated samples (G2) (p-value < 0.05). p16INK4a and E6-transcript positivity were found in 57% and 24% of the HPV positive samples, respectively. The OS (overall survival) showed a not statistically significant difference in prognosis between HPV positive sand negatives (10, 47.6%, vs. 4, 44.4%; p-value = 0.25). Conclusions: HPV-DNA and p16INK4a positivity confirmed the role of HPV in anal carcinoma. Our findings could support the implementation and scale-up of HPV vaccination in males and females to decrease the incidence of HPV-associated cancers. Further studies are needed to better clarify the prognostic role of HPV/p16 status.