The present study investigated the effect of varying osmolarity of tris egg yolk citrate glycerol extender on seminal attributes of buffalo semen at various hours of refrigeration at 5 ºC. Twenty four ejaculates having mass motility ≥ 3+ from 4 bulls (6 ejaculate from each bull) were collected. Each ejaculate was divided into four group’s viz., Groups I, II, III and IV, diluted with extenders having osmolarities of 240, 260, 280 and 300 milliosmol/kg (mOs/kg), respectively, to 80×106 sperm/ml. After dilution, semen samples were filled in French mini straws and kept at 5°C and evaluated at 0, 24, 48 and 72 hours for various seminal attributes such as individual motility, viability, acrosomal integrity, and hypo-osmotic swelling test. No significant difference in individual motility at 0 hour was observed among all the groups. However at 24, 48 and 72 hours, the individual motility was significantly (P<0.05) higher in groups I and II as compared to groups III and IV. Other seminal attributes such as viability, acrosomal integrity and hypo-osmotic swelling test were significantly (P<0.05) higher in group II (260 mOsm/kg) as compared to other groups. It is concluded that seminal characteristics such as individual motility, viability, hypo-osmotic swelling (HOS) test and acrosomal integrity were significantly affected when the osmolarity of the extender was changed from 260 to 240, 280, and 300 mOsm/kg.