We investigated the cryopreservation protocol for sex sorted water buffalo spermatozoa. Sex-sorting of spermatozoa was carried out in three different techniques i.e. modified swim-up, paramagnetic nanoparticles and differential pH. After sorting, sperm cryopreservation was carried out. To optimize the sex sorted sperm cryopreservation technique, we tested three different cryodiluents. Briefly, the harvested sex-sorted spermatozoa were further divided into three parts and diluted in three different cryodiluents i.e. 1) tris-citric acid (TCA; Tris 3.0 g/100 mL, Citric acid 1.56 g/100 mL, Fructose 0.2 g/100 mL, Streptomycin sulfate 0.01 g/100 mL, fresh chicken egg yolk 20 mL, glycerol 7 mL, Distilled water 73 mL), 2) skimmed milk (SM; Skimmed milk 1 g/100 mL, Egg yolk 5 mL, Streptomycin sulfate 0.01 g/100 mL, Glycerol 7mL), and 3) commercial Triladyl® (TDL) to final concentration of 4 × 106 spermatozoa/ml respectively. Diluted samples were cooled to 4 oC in 120 min and equilibrated at 4oC for 4 hours, packaged in 0.54 mL French straws, frozen from initial holding at +4°C for 2 min, from +4°C to -20°C at 10 ºCmin-1, from -20°C to -100°C at 30°C min-1 and final holding for 1 min at -100°C with programmable freezer and stored in liquid nitrogen at -196oC. Post- thaw analysis was performed 24 hours after freezing. Wherein, the total motility (TM), progressive motility (PM), rapid velocity (RV) plasma membrane integrity (PMI), viability along with acrosomal integrity (VIA/IACR) of spermatozoa were determined. At postthawing, results of TM (78.55±2.02), PM (31.83±1.85), RV (34.00±3.49), PMI (60.12±2.92) and VIA/IACR (58.84±1.93) were significantly higher in TDL as compared to other groups. In conclusion, Triladyl® is the extender of choice for the cryopreservation of buffalo sexed spermatozoa.