Sperm thermotaxis, the directed movement of sperm along temperature gradients, helps spermatozoa to navigate the female reproductive tract. Although this phenomenon has been studied in many species, its relevance in livestock, particularly in rams, remains to be explored. The present study aimed to determine in vitro thermotactic behaviour of ram spermatozoa and its impact on sperm concentration, motility and viability across different temperature gradients. Sperm collected from a mature Awassi ram was subjected to a swim-up procedure and incubated for 60 min in two thermal gradient tubes consisting of two compartments maintained at 33 °C and 41 °C, respectively. An isothermal tube with two compartments maintained at 38 °C served as the control. Sperm concentration, kinetic parameters, membrane integrity, and viability were assessed in each compartment. No significant differences were observed between the compartments of the isothermal control. In contrast, sperm concentration was significantly higher in the cooler compartment (33 °C) than in the warmer (41 °C) compartment of the thermal gradient group. Motility also tended to be higher at 33 °C (P = 0.054). Although some velocity parameters differed numerically between temperature conditions, these differences were not statistically significant. Exposure to 41 °C markedly reduced sperm viability compared with both the initial semen sample and the cooler compartment. These findings demonstrate that ram spermatozoa show thermotactic behaviour in vitro and preferentially accumulate in cooler regions. This response is also associated with improved motility, membrane stability and viability. These findings suggest that thermotaxis may contribute not only to sperm navigation but also to the selection of spermatozoa with higher motility and viability in small ruminants.