Background One third to one half of penile squamous cell carcinomas (SCCs) are related to human papillomavirus (HPV) infection. Viral detection is usually carried out by polymerase chain reaction (PCR) or other molecular methods. In this study, we evaluated p16INK4a immunohistochemical expression, which is simpler and less costly, as a potential marker of high-risk HPV (HR-HPV) infection in penile SCC. Design and Methods We pathologically classified 202 invasive penile carcinomas and performed HPV genotyping by short PCR fragment (SPF)10 PCR and p16INK4a immunohistochemistry. We also evaluated HPV and p16INK4a according to the histologic subtypes of penile SCC. Tumors depicting continuous p16INK4a immunostain in all neoplastic cells were considered positive. HPV and p16INK4a status were compared using classifier performances and concordance indexes. Results Evidence of HPV (low-risk and high-risk genotypes) was found in 63 cases (31%) by PCR. Fifty-three p16INK4a-positive cases were identified (26%). Overexpression of p16INK4a had a sensitivity of 67% and a specificity of 91% for defining the HPV status. Concordance indexes between p16INK4a and HPV status were high (≥78%) in general cases and in all histologic subtypes of penile SCC. The stain was useful in the differential diagnosis of basaloid and low-grade warty carcinomas. Low-risk HPV genotypes were found in 5 tumors, 4 of which were p16INK4a negative. Basaloid and nonbasaloid high-grade (grade 3) SCCs were more likely to be HR-HPV positive when compared with grades 1 to 2 tumors (PINK4a overexpression was found to be a reliable marker for HR-HPV and a helpful tool in the differential diagnosis of low-grade verruciform and high-grade solid penile tumors. SCC variants depicting basaloid features were more likely to be HPV and p16INK4a positive than low-grade, keratinizing lesions. We also observed a tendency toward HPV positivity in high-grade nonbasaloid tumors. Our results indicated a concordance between HPV and p16INK4a status and this observation may have diagnostic and prognostic implications.