Objectives Penile swab sampling is the method of choice when testing for human papillomavirus (HPV) in men. Urine sampling is already used in routine sexually transmitted infections (STI) diagnostics and could provide a less invasive sampling method in men to detect HPV. Therefore we compared detection of HPV types in urine samples and penile swabs by the highly sensitive SPF10-LiPA25 system. Methods First void urine and self-obtained penile swab samples were collected from 120 men, with a mean age of 29.4 years, visiting a STI clinic in South Limburg, the Netherlands. In total 111 of 120 men were included in the analysis. Broad-spectrum HPV DNA amplification and mucosal HPV genotyping were performed using the SPF10 DEIA-LiPA25 system (SPF10 HPV LiPA, V.1). Results In total 75 (68%) men were positive for HPV in the combined analysis. Sixty-six (59%) paired samples were concordant in being positive or negative. In 39% of the men HPV DNA was detected only in the penile swab. In 2% of the men HPV DNA was detected only in the urine sample. Considering penile swabs as the gold standard, a sensitivity of 41% (95% CI 30% to 53%) and a specificity of 95% (95% CI 81% to 99%) was found. In 6 (5%) urines high risk types were repeatedly found that were not detected in the matching swab. Conclusions Urine samples are not comparable to penile swabs in the detection of HPV in men. However, the addition of urine samples to penile swabs could be of use in epidemiological or clearance studies.