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Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3 + lymphocytes, γδ T-cells, CD8 + γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4 + and CD8 + T-cells, and B-cells than calf lymphocytes. The proportion of CD4 + T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle., (Copyright © 2022 Elsevier B.V. All rights reserved.)