Background: We examined sperm quality in a cohort of city policemen in the capital city of Prague, an area experiencing elevated air pollution levels as a result of a substantial rise in traffic. As the control group, policemen working in Ceske Budejovice were selected due to this city’s location in a nonindustrial area characterized by substantially lower pollution levels compared to Prague. Methods: Semen samples from 63 Prague and 16 Ceske Budejovice city policemen were examined for standard semen parameters, sperm motion characteristics and sperm DNA fragmentation. Average daily air pollutant concentrations were recorded by stationary monitoring for 90 days preceding the collection of semen samples. Results: Sperm volume, concentration, DNA fragmentation index and the percentage of immature sperm in Prague policemen did not differ significantly between the monitoring periods. The percentage of total and progressive motility, % vitality, % normal sperm morphology and % acrosome-intact sperm were significantly lower in autumn. In policemen from Ceske Budejovice, there were no significant differences in any of the evaluated parameters of semen quality between the monitoring periods. During both spring and autumn samplings, a significant decrease in sperm volume was observed in the policemen from Prague compared to Ceske Budejovice. In autumn, significantly lower values of sperm viability percentage and motility were detected in Prague. Conclusion: City policemen working in a large city with high air pollution levels exhibited significantly poorer sperm quality parameters than policemen from a smaller city with lower traffic intensity. This difference was observed in the summer months