Ovum pick (OPU) up is a technique upon which in vitro embryo production (IVP) depends. It permits donor cow maternal ancestry to be assessed by mtDNA analysis. Repetitive ablation of follicles is thought to interfere with the donor cow endocrine profile and influence embryo yield. The objective was to evaluate the effects of mtDNA and OPU order on IVP fertility traits. Gyr donors (85) were submitted to 363 OPU sessions (5 OPU sessions/donor). Donor mtDNA was extracted from leukocytes and sorted by the presence of the HindIII restriction site within the amplified region, indicative of Bos taurus taurus mtDNA (Paneto et al. 2008 Genet. Mol. Res. 7, 592). All animals in the donor pedigree were classified, and the population was divided into two groups according to their maternal genetic grouping: Bos taurus indicus and Bos taurus taurus. For statistical analyses, data from 5 OPU sessions per each donor were submitted to the mixed model procedure of SAS® (SAS Institute Inc., Cary, NC, USA), using the lowest Akaike value to determine the best covariance structure between repeated OPU session results. The model included the effect of OPU session order (1–5) and mtDNA-based maternal grouping (Bos taurus v. Bos indicus) as independent variables. Total and viable oocytes, as well as blastocyst yield per OPU session, were the dependent variables studied. The means of total and viable oocytes and blastocysts produced per donor per OPU were compared by the Tukey test, at 5% significance. The combined OPU sessions resulted in 6084 oocytes, 2537 embryos, which produced 1105 pregnancies. Mean numbers (n = 42 donors/OPU session) of total and viable oocytes between OPU sessions 1 (31.9 ± 4.6 and 19.2 ± 2.9), 2 (35.3 ± 3.5 and 21.2 ± 2.2), 3 (28.9 ± 3.7 and 19.3 ± 2.4), 4 (25.0 ± 5.2 and 18.9 ± 33), and 5 (20.5 ± 6.3 and 13.8 ± 4.0) did not differ. Mean blastocyst production after IVP between OPU sessions 1 (6.5 ± 1.4), 2 (5.15 ± 1.04), 3 (5.5 ± 1.1), 4 (5.8 ± 1.6), and 5 (5.2 ± 1.9) was similar. Mean viable oocyte number was greater (P < 0.0001) for B. t. taurus (21.7 ± 1.4, n = 192 OPU sessions) compared with B. t. indicus (16.1 ± 0.9, n = 138 OPU sessions) maternal genetic groupings. However, in vitro blastocyst yields were similar (P = 0.23) between maternal genetic groupings (7.3 ± 1.6 and 6.2 ± 1.4 for B. taurus and B. indicus, respectively). In conclusion, repeated OPU sessions did not reduce oocyte and embryo yields as expected. Maternal B. taurus genetic origin was associated with higher oocyte quality, although it was not translated into higher embryo yields after in vitro culture. Results warrant further research, which may result in additional selection criteria for OPU Gyr donors considering their maternal genetic background.