Objective: The aim of this study was to evaluate the effect of vitrification with two methods of 4 and 2-step dehydration on sheep ovarian tissue. Materials and Methods: Sheep ovarian tissue samples were collected and allocated to three groups of fresh 4-step vitrified and 2-step vitrified. A modified carrier and vitrification solution was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in vitrified-warmed tissues. The base medium (BM) containing hepessed tissue culture medium (HTCM) supplemented with 10% human serum albumin (HSA) was used as the solvents for vitrification and warming solutions. The ovarian tissue pieces were dehydrated by using regimens; 4-step: (1) 3.75% ethylene glycol (EG) and dimethyl sulfoxide (DMSO); (2) 7.5 % EG and DMSO; (3) 10% EG and DMSO; (4) 15% EG and DMSO + 0.25 mM sucrose in the base medium each for 5 minutes at 4°C and 2-step: (1) 7.5% EG and DMSO; (2) 15% EG and DMSO + 0.25 mM sucrose in the base medium each for 10 minutes at 4°C. Also imaging of apoptotic follicles was performed in fresh and vitrified tissues using TUNEL protocol. Results: The proportion of intact antral follicles in the fresh (66.66% ± 0.33) and 2-step vitrified (56.66% ± 0.16) groups were significantly higher than that in the 4-step vitrified (0%) group whereas the difference of this proportion between fresh and 2-step vitrified was not significant But between the three groups in the proportion of primordial primary and preantral follicles the differences was not statistically significant. The apoptosis imaging of ovarian pieces also did not show statistically differences between all the groups. Conclusion: These results indicated that sheep ovarian tissue vitrification by 2-step method is simpler and more effective than those of 4-step method. On the other hand as sheep and human ovarian tissue are more similar this technique can be exam for cryopreservation of the human ovarian tissue too.