Purpose <p>To investigate the potential role of human papillomavirus (HPV) in early onset prostate cancer (PCa) through HPV-DNA detection and genotyping, and immunohistochemical evaluation of p16^INK4a expression.</p> Methods <p>This retrospective cross-sectional study included patients diagnosed with PCa at ≤ 55&#xa0;years of age between 2010 and 2021. Patients with a familial history of PCa, incomplete medical records, immunodeficiency, or insufficient tissue for molecular analysis were excluded. Formalin-fixed, paraffin-embedded (FFPE) prostate biopsy samples were analyzed for HPV-DNA using the HPV 3.5 LCD-Array Kit (Chipron GmbH, Berlin, Germany), a multiplex PCR-based assay targeting 32 HPV genotypes. Immunohistochemical staining for p16^INK4a was performed to assess its expression. Clinical data, including PSA levels, Gleason score, ISUP grade, and EAU risk group, were compared between HPV-positive and HPV-negative cases.</p> Results <p>HPV-DNA was detected in 12 of 45 cases (26.7%), with genotype 62 being the most frequent. Multiple HPV genotypes were identified in three cases (6.7%). p16^INK4a immunopositivity was observed in 91.7% of HPV-positive tumors compared with 69.7% of HPV-negative tumors (<i>p</i> = 0.240). HPV-positive patients showed higher ISUP grades than HPV-negative patients (<i>p</i> = 0.029) and were more likely to be in intermediate or high-risk groups (<i>p</i> = 0.028). No significant differences were observed in PSA levels or Gleason scores.</p> Conclusion <p>The detection of HPV-DNA in a considerable proportion of early onset PCa specimens, together with the more frequent but not statistically significant p16^INK4a positivity in HPV-positive tumors, suggests a possible association with prostate tumor biology. However, the presence of low-risk HPV genotypes and the non-specific nature of p16^INK4a expression complicate the interpretation of a direct oncogenic role.</p>