The aim of the current study was to instead of egg yolk with various levels of coconut water as a cryoprotectant in Tris-extenders and to investigate its effect on quality and fertility of frozen buffalo bulls' semen. Semen was collected twice a week from five mature buffalo bulls for 12 weeks and ejaculates with 70% progressive motility prior were pooled to have sufficient semen volume for a replicate and to eliminate the bull effect. After evaluation semen quality, the pooled fresh semen was divided into five equal fractions; one fraction was diluted with Tris-egg yolk extender (control) and the other four replects, whole egg yolk was replaced by different concentrations of five; 10; 15 and 20% coconut water extracted. Semen was diluted to eighty million sperm/ml packaged into 0.25ml straws, cooled held at 5ºC for4 h, and then frozen in liquid nitrogen and stored at –196ºC until for artificial insemination. The percentages of sperm progressive motility, live sperm, sperm abnormality, intact sperm acrosome and plasma membrane integrity were evaluated post dilution, post–equilibration and post frozen–thawed processes.  The results revealed that 20% coconut water extender was more effective on cryopreservation of semen characteristics (progressive motility, live sperm, sperm abnormality, intact sperm acrosome and plasma membrane integrity) of buffalo spermatozoa than whole egg yolk extender and other coconut milk extenders. Also, conception rates were higher in 20% coconut water extender compared with control egg yolk extender (66.7% vs 58.3%, respectively). In conclusion, added20% coconut water to extender improves the freezability and fertility of buffalo spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo bulls semen.