This study was performed to determine the associations between human papillomavirus (HPV) infection and cytological changes in the male glans and urethral samples. Two rubbed samples of the glans and distal urethra were collected from 160 patients with urethritis, and the collected cells were placed into preservative solution for liquid-based cytology. DNA was extracted from all samples, and β-globin gene amplification, HPV-DNA test, and HPV genotyping were performed. After papanicolaou staining of each sample, cytological findings were assessed based on nine non-classic signs, and compared with the results of HPV-DNA test. Among the β-globin positive samples, HPV (any type) was detected in 33 and 25% of the glans and urethra samples, respectively. High-risk HPV-DNA was detected in 25 and 15% of the glans and urethra samples, respectively. In the glans samples, cytological signs of HPV infection were observed in 17% of low-risk HPV-positive samples and in 58% of high-risk HPV-positive samples. Cytological atypia suspected to indicate penile intraepithelial neoplasia were observed in 17 and 33%, respectively. On the other hand, abnormal cells were observed in 29% of low-risk HPV-positive and 83% of high-risk HPV-positive urethral samples. Cytological atypia suspected to be urethral intraepithelial neoplasia was observed in 29 and 65%, respectively. In situ hybridization demonstrated the presence of HPV-DNA in the morphologically abnormal cells in 31 and 35% of high-risk HPV-positive glans and urethral samples, respectively. Cytological changes similar to cervical intraepithelial neoplasia in females could be detected in the HPV-positive glans and urethral samples. Diagn. Cytopathol. 2014;42:491–497. © 2013 Wiley Periodicals, Inc.