Abstract: The study aimed to investigate the effects of vitrification on development of donkey oocytes. Fresh ovarian tissues were collected and the isolated oocytes were treated in the following three ways: direct freezing (T1 group), recovery after cryopreservation + in vitro maturation treatment for 20 h (T2 group) or 40 h (T3 group). At the same time, fresh GV oocytes were included and served as the corresponding control groups: fresh cells without maturation treatment (C1 group) and those matured for 20 h (C2 group) or 40 h (C3 group). The cells treated in different ways were subjected to transcriptome sequencing. The obtained original data were filtered by Trimmomatic (v0.36) software, and the subsequent data were analyzed. After filtering, the original data were compared with the reference gene pool for annotation analysis and methylation site recognition, and the differences in methylation level between the different groups were compared. The results showed that the methylation levels of T1 and T2 groups were higher than those of C1 and C2 groups, respectively, and the methylation level of T3 group was lower than that of C3 group. In the oocytes treated with vitrification, the highest methylation level of CpGs was observed T1 group, followed by T2 group and T3 group. A total of 1 269 differentially expressed genes were selected, and 677 of them were found to have expressional differences between cryopreserved and unfrozen cells. The most significant differences in methylation level before and after vitrified cryopreservation in donkey oocytes were observed in GFM1 and MRPL15 genes, and the gene pathway was enriched in the mitochondrial extension pathway, which resulted in the lower maturation rate of oocytes after vitrified cryopreservation. The results of this study laid experimental basis for determining the optimal conditions for oocytes cryopreservation.