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The aim of this study was to determine the effect of seasonality on post‐thaw sperm quality in Prochilodus lineatus. Sperm from 43 males was cryopreserved throughout the reproductive season (November–March). Fresh sperm quality, concentration, volume, seminal plasma pH, osmolality and ion concentration were analysed. Post‐thaw sperm was analysed for its motility rate, velocities, beat‐cross frequency, cell morphology, oxidative stress and fertilization capacity. Prochilodus lineatus fresh sperm yielded high‐quality during the reproductive period (motility >80%). Post‐thaw sperm quality changed throughout the season, with higher motility (63.2%–72.3%) and curvilinear velocity (VCL) (55.9–59.2 µm/s) from December to March. Sperm concentration was negatively correlated with post‐thaw sperm motility and VCL, and positively correlated with seminal plasma Ca2+ concentration, catalase activity (CAT) and reactive oxygen species (ROS). Seminal plasma Ca2+ was positively correlated with ROS and negatively correlated with post‐thaw sperm motility. Post‐thaw sperm CAT and fertility correlated positively. Although post‐thaw sperm quality was reduced in November, the increased CAT was efficient in maintaining its fertilization capacity. In order to face seasonal influence, the optimal period to cryopreserve P. lineatus spermatozoa is from December to March, when sperm exhibits characteristics which make spermatozoa more prone to resist cryodamage.