Background <p>Spermatogenesis is a temperature-sensitive process, but previous studies on seasonal variation in semen quality have produced conflicting results, often due to differences in sample sizes, methodologies, or local climates. Most studies have reported peak sperm motility in colder months, although findings vary by country. This study investigated whether the quality of semen (defined here by sperm motilities and concentrations) varies seasonally in Denmark and Florida, and whether outdoor ambient temperatures contributed to any seasonal trends.</p> Design <p>This retrospective observational study included data from 15,581 men applying to be sperm donors at Cryos International from 2018 to 2024 at two general locations: 10,670 men from four Danish cities (Aarhus, Aalborg, Odense, and Copenhagen) and 4,911 men from Orlando, Florida. These men were 18–45&#xa0;years old and resided near the collection sites. All ejaculates were analysed within one hour of collection using the same computer-assisted semen analysis (CASA) system and standardised temperature-controlled protocols to quantify ejaculate volume, sperm concentration and the concentrations and counts of progressively motile sperm (grades a and b) in each ejaculate. The effects of outdoor temperatures on sperm motility during the month of collection and two months earlier (representing early spermatogenesis) were also modelled. We used nonlinear statistical methods (GAMs) to accurately analyse and display the month-to-month seasonal variation, while controlling for and illustrating the nonlinear effects of male age, monthly average temperatures, and the longitudinal trends during the study period (2018–2024) on those semen parameters.</p> Results <p>Strong and consistent seasonal variation was observed in the concentrations of progressively motile (grades a and b) sperm in both Denmark and Florida. The concentration of rapidly progressive (grade a) sperm was highest in the warmer months (June-July) and lowest in the colder months (December-January) in both countries, despite climatic and demographic differences. There was no evidence of seasonal variation in ejaculate volume or total sperm concentration, indicating that the rate of spermatogenesis did not vary seasonally. The number of progressively motile sperm (TMSC) in an ejaculate also varied seasonally, even after controlling for monthly average outdoor temperature in the contemporaneous and preceding two months, suggesting that other seasonal factors, possibly related to lifestyle or environment, may affect sperm motility.</p> Conclusions <p>Semen quality (measured as the concentration and number of progressively motile sperm) exhibits clear and consistent seasonal variation in the male populations living in both a temperate and a subtropical climate. These findings highlight the importance of accounting for seasonality when assessing semen parameters for fertility evaluation and indicate that there is seasonal variation in semen quality, even in warm climates.</p>