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Objective Mitochondria are an excellent source for explaining cytoplasmic inheritance because of their important roles in cell metabolism and because they are primarily inherited maternaly in animals. In domestic animals, various studies have reported an association between mitochondrial DNA polymorphism and production and reproduction traits. The aim of this study was to investigate the polymorphism of ATPase6 and ATPase8 genes and their effects on the rates of in vitro maturation of oocytes (IVM) and in vitro culture of embryos (IVC) in Sanjabi sheep. Materials and methods Blood and ovary samples of adult Sanjabi ewes were collected from Bistoon slaughterhouse located in Kermanshah. In the laboratory, after separating the excess tissue around the ovaries and washing them, a cumulus-oocyte complex with a diameter of more than 3 mm from the follicles was aspirated. 100 samples containing more than two oocytes were transferred to dishes containing in vitro maturation (IVM) medium. After the oocytes matured, fresh Sanjabi ram sperm were used for in vitro fertilization of each sample. For in vitro culture, putative zygotes formed in the previous stage were cultured in synthesized ovarian fluid medium for 48 hours. DNA extraction was performed using the salting out method. A 895 bp fragments of DNA containing ATPase6 and ATPase8 genes were amplified by PCR and the samples were genotyped using a modified SSCP method. The Kruskal-Wallis nonparametric test was used to investigate the relationship between the observed genotype patterns and IVM and IVC rates. Results The results of this study showed the existence of polymorphisms in the studied gene regions and five different band patterns A (50%), B (12%), C (9), D (21) and E (8%) were observed. Analysis of the relationship between these patterns and IVM and IVC showed no significant relationship between them (p>0.05). Conclusions Despite the observation of polymorphisms in ATPase6 and ATPase8 genes using the modified SSCP method, these polymorphisms had no effect on the rate of IVM and IVC.