Oocytes cryopreservation in mammalian species has gained a rapid pace during the past couple of decades emphasizing its importance in various assisted reproductive technologies .Aim of this work was to study effects of different cryoprprotectant agents: Ethylene Glycol (EG), Dimethyl sulfoxide (DMSO) and combination of them (EG+DMSO) on the viability, mitochondrial distribution and intensity of in vitro matured vitrified/ thawed buffalo oocytes by determine: 1) the effect of different cryoprotectants on the viability and developmental competence of vitrified/thawed in vitro matured buffalo oocytes. 2) The effect of different cryoprotectants on the mitochondria distribution and intensity of vitrified/thawed in vitro matured buffalo oocytes. Ovaries were collected from EL-Warak slaughter house, Cairo, Egypt. Excellent and good oocytes were in vitro matured in TCM-199 +IGF for 22 h in incubator at 38.5 ₒC in 5% CO2 and humidified atmosphere. Morphologically normal matured oocytes with first polar were placed in equilibration solution (VS1) for 1 min then oocytes were transferred to (VS2) for 30 sec. VS1 is half concentration of VS2 which contain EG 40% (EG group) or DMSO 40% (DMSO group) or EG 20%+DMSO 20% (Combination group). Oocytes were loaded in sterile 0.25 ml straws and stores in liquid nitrogen for 7-10 days. Morphological changes (normal and abnormal) of vitrified thawed in vitro mature buffalo oocyte were detected under invertd microscope. Mitotracker red stain and confocal microscope (Zeiss 710) used to study the mitochondrial distribution and Hochest dye to study the viability of in vitro vitrified/ thawed matured buffalo oocytes. The obtained results revealed that using a combination of EG+DMSO for vitrification resulted in the best quality of in vitro vitrified thawed buffalo oocytes which was demonstrated by significantly higher percent of morphologically normal recovered oocytes and transferable embryos when compared with EG and DMSO. More mitochondria were diffusely distributed in the fresh oocytes (93.33%) than in all vitrified groups oocytes: DMSO group (63.33%), EG group (79.99%) and DMSO+ EG group (79.99%). The Mean No. of mitochondrial intensity of recovered mature oocytes vitrified in EG+DMSO group (186.22) was significantly higher (P < 0.05) than those vitrified in EG group (153.33) and DMSO group (146.98). In conclusion: Combination of EG+DMSO cryoprotectants improve the viability and developmental competence of in vitro matured vitrified/thawed buffalo oocytes.