Mitochondria have a broad range of functions that affect reproduction, and structural as well as quantitative variation in mtDNA has been associated with gamete quality and reproductive success. To investigate the mitochondria effect on in vitro embryo production, we collected oocytes by ultrasound-guided follicular aspiration from donor cows known to differ in the developmental capacity, measured by the blastocyst formation rate, of their oocytes. To evaluate the potential effects of mtDNA and mitochondrial function on oocyte quality, the donor cows' mtDNA control region was sequenced and, after pairwise comparisons of polymorphisms, animals were grouped into two major haplogroups. The number of mtDNA molecules per oocyte was quantified by real-time PCR, and the adenosine triphosphate (ATP) content was measured in each oocyte to identify variations between haplogroups. Overall, ATP stocks in oocytes of the two haplogroups differed significantly (P < 0.05; means ± SEM) both at the germinal vesicle and metaphase II stages (2.8 ± 0.06 pmol vs. 2.6 ± 0.07 pmol and 2.9 ± 0.1 pmol vs. 2.3 ± 0.06 pmol, respectively). The proportion of development to blastocyst was significantly different between haplogroups (22.3 ± 2.1 % vs. 36.7 ± 2.9 %). The number of mtDNA molecules per oocyte was highly variable (377 327 ± 14 104, ranging from 2.0 × 10³ to 1.2 × 10⁶) but not significantly different between the two haplogroups; significant differences were observed between animals without any apparent relationship to blastocyst production. These data suggest that mitochondria and mtDNA haplogroup affect the developmental capacity of bovine oocytes in vitro.