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Human papillomavirus (HPV) has been reported in 12-82% of penile squamous cell carcinomas (SCC). There is an association of the virus with basaloid and warty carcinomas but the reported prevalence is variable. The causes of these variations are not clear. They may be owing to geographic differences, the use of different techniques to detect HPV, the status of the original paraffin blocks, or to variable criteria in tumor classification. The aims of the study were to determine the prevalence of HPV in penile SCC and subtypes using a sensitive technique, to investigate genotypes involved, and to search for other morphologic features associated with the virus from a series of cases from Paraguay. HPV detection was done by SPF-10 polymerase chain reaction followed by DNA enzyme-immunoassay and genotyping by LIPA 25 (version 1). Samples were tested at Catalan Institute of Oncology, Barcelona, and cross testing was carried out at the Delft Diagnostic Laboratories in The Netherlands. HPV was detected in 64 of 202 cases (32%). Thirteen tumors had multiple HPV genotypes. Most prevalent genotypes were HPV-16 (46 cases), HPV-6 (6 cases), and HPV-18 (4 cases), either in single or in multiple infections. HPV was preferentially associated with warty-basaloid (82%), basaloid (76%), and warty (39%) carcinomas and not detected in verrucous, mixed verrucous-papillary, pseudohyperplastic, and pseudoglandular SCCs. There was a strong association between HPV and higher histologic grade. Basaloid cells were more frequently found in HPV positive tumors (72%) and this association was statistically significant in univariate and multivariate analyses. Cells with koilocytotic features and keratinizing squamous cells were also present but to a much lesser degree (47% and 19%, respectively). In summary, HPV was found in a third of the cases and the most common genotype was HPV-16. Low-risk genotypes were rarely found in single infections, representing 4 cases among all analyzed (2%). There was an association between HPV presence and higher histologic grade and with basaloid, warty-basaloid, and warty carcinomas. Our results also suggest that, in penile SCC, the basaloid cell is the best tissue marker for oncogenic HPV infection.