Mitochondria are central to metabolism and are the primary energy producers for all biosynthesis. The objective of this study was to compare mitochondrial enzyme activities of high- and low-producing dairy cows in early lactation to genetic markers of lactation performance. Blood samples were collected from 56 Holstein cows (70 ± 11 DIM) and mitochondria isolated from peripheral blood mononuclear cells (PBMC). Mitochondrial function was assessed by measuring the activity rates of citrate synthase (CS), complex I (CI), complex IV (CIV), and complex V (CV). Milk samples were collected 9 times within a week of blood collection and analyzed for major components using a MilkoScan FT2 by FOSS. Data were analyzed using GLM and the Mixed procedure of SAS (version 9.4, SAS Inst. Inc., Cary, NC) for high or low groups for each production parameter with cow as the experimental unit of interest and dependent variables parity, and DIM as covariates. There were no interactions between milk yield level (high or low) and parity. Complex I and CV activities were lower in low producing cows than high producing cows for both the multiparous and primiparous groups (P ≤ 0.04) and CV was not influenced by parity for both multiparous and primiparous cows (P > 0.1) across production parameters (ECM, milk fat, and total solids) suggesting that it may serve as a marker of a cow’s ability to produce regardless of parity. Genetic indices of fluid merit, milk and protein were not correlated with CS, CI, CIV and CV activities (P > 0.1). The genetic index for fat was positively associated with CI activity for primiparous cows (P ≤ 0.02, R² = 0.27). Comparisons of genetic predictors for milk fat to actual fat yield had positive correlations and were observed only in primiparous cows (P ≤ 0.01, R² = 0.40) while multiparous cows showed no associations (P > 0.1, R² = 0.05) suggesting that genetic markers alone may not accurately predict fat production beyond the first lactation and that the use of mitochondrial enzyme activities may provide a better index of cow production potential as it considers both the animal’s ability to process nutrients and parity related changes.