Mitochondrial DNA copy number (mtDNA CN) has been shown to be highly heritable and associated with traits of interest in humans. However, studies are lacking in the literature for livestock species such as beef cattle. In this study, 2,371 individuals from a crossbred beef population comprising the Germplasm Evaluation program from the U.S. Meat Animal Research Center had samples of blood, leucocyte, or semen collected for low-pass sequencing (LPS) that resulted in both nuclear DNA (nuDNA) and mitochondrial DNA (mtDNA) sequence reads. Mitochondrial DNA CN was estimated based on the ratio of mtDNA to nuDNA coverages. Genetic parameters for mtDNA CN were estimated from an animal model based on a genomic relationship matrix (~87K SNP from the nuDNA). Different models were used to test the effects of tissue, sex, age at sample collection, heterosis, and breed composition. Maternal effects, assessed by fitting a maternal additive component and by fitting eleven SNP on the mtDNA, were also obtained. As previously reported, mtDNA haplotypes were used to classify individuals into Taurine haplogroups (T1, T2, T3/T4, and T5). Estimates of heritability when fitting fixed effects in addition to the intercept were moderate, ranging from 0.11 to 0.31 depending on the model. From a model ignoring contemporary group, semen samples had the lowest mtDNA CN, as expected, followed by blood and leucocyte samples (P ≤ 0.001). The effect of sex and the linear and quadratic effects of age were significant (P ≤ 0.02) depending on the model. When significant, females had greater mtDNA CN than males. The effects of heterosis and maternal heterosis were not significant (P ≥ 0.47). The estimates of maternal and mtDNA heritability were near zero (≤0.03). Most of the samples (98%) were classified as haplogroup T3. Variation was observed in the mtDNA within Taurine haplogroups, which enabled the identification of 24 haplotypes. These results suggest that mtDNA CN is under nuclear genetic control and would respond favorably to selection.