This study was designed to determine the effect of different osmotic pressures on spermatozoa characteristics for cryopreservation of buffalo bull semen using tris egg yolk extender (TEYE). Semen of Nili-Ravi buffalo bulls (n = 04) housed at Semen Production Unit, Qadirabad, Sahiwal, Pakistan, was collected at weekly interval for 10 weeks. Three solutions of tris-citric acid-fructose with osmotic pressures of 255, 275, and 295 mOsm/kg were used in extender preparation. Semen straws containing 20x10⁶ spermatozoa were processed and stored at −196°C using liquid nitrogen. Post thaw analyses of spermatozoa included motility, viability, acrosomal integrity, plasma membrane integrity, DNA integrity and lipid per-oxidation. One-way ANOVA was implied and in case of significant differences among groups, the Duncan’s Multiple Range Test was applied and results were considered significant at P < 0.05. Spermatozoa motility, acrosomal and DNA integrity were significantly (P < 0.05) affected by varying osmotic pressures of the solutions. Higher rate of spermatozoa motility, acrosomal and DNA integrity were recorded at osmotic pressure 275 and 295 mOsm/kg compared with 255 mOsm/kg. However, rate of spermatozoa viability, plasma membrane integrity and lipid per-oxidation was not affected at any of the osmotic pressure treatments. It is concluded that consideration of osmotic pressure of the solution used in extender preparation may be useful in improving the quality of cryopreserved buffalo bull semen.