Background:Sperm motility parameters, which can be measured objectively and repeatedly by a computer-assisted sperm analysis (CASA) system, are important indicators of sperm quality. However, the sperm motility parameters assessed by a CASA system can be affected by various factors, including instrument components and settings, sperm preparation or analysis procedures. To date, no standardized protocol is available that would permit to assess sperm kinetic characteristics in passerine birds and this lack precludes any comparison of sperm swimming ability and sperm quality across species. Methods: In this study, we chose the Tree Sparrow (Passer montanus) as the object to evaluate sperm motility param-eters, including sperm motility, sperm velocity and sperm movement trajectory, at different analysis time, tempera-tures and pH using the WLJY-9000 CASA system. Results: Sperm motility parameters remained statistically unchanged at 1-9 min. Progressive motility was similar at 38 ℃ and 40 ℃, but a greater percentage of slow progressive sperm was detected at 38 ℃ compared to 40 ℃ and 42 ℃. Additionally, progressive motility was lower and immotility was higher at 42 ℃ than 38 ℃ and/or 40 ℃ (close to the body temperature of the Tree Sparrow). The percentages of rapid progressive sperm, progressive sperm and immotile sperm were statistically similar at pH 7.0, 7.5 and 8.0 with the exception of lower percentage of progressive sperm at pH 7.0 compared to pH 7.5. In addition, slower sperm velocity and worse sperm movement trajectory were found at pH 6.0 and 9.0 than those at pH 8.0, 7.5 or 7.0. Conclusions: Our study indicates that the ideal conditions for sperm motility parameters assessment in Tree Spar-row are obtained between 1 and 9 min after dilution, an environment at body temperature (40 ℃) and a pH around 7.5-8.0. The results of this study provide a reference for the evaluation of sperm characteristics and sperm quality using a CASA system in passerine birds.