Immune cells resident in adipose tissue have important functions in local and systemic metabolic homeostasis. Nevertheless, these immune cell populations remain poorly characterized in bovines. Recently, we described diverse lymphocyte subpopulations in adipose tissue of Holstein-Friesian cows. Here, we aimed at characterising myeloid cell populations present in bovine adipose tissue using multicolour flow cytometry, cell sorting and histochemistry/immunohistochemistry. Macrophages, CD14+CD11b+MHC-II+CD45+ cells, were identified in mesenteric and subcutaneous adipose tissue, though at higher proportions in the latter. Mast cells, identified as SSC-AhighCD11b−/+CD14−MHC-II−CH138A−CD45+ cells, were also observed in adipose tissue and found at higher proportions than macrophages in mesenteric adipose tissue. Neutrophils, presenting a CH138A+CD11b+ phenotype, were also detected in mesenteric and subcutaneous adipose tissue, however, at much lower frequencies than in the blood. Our gating strategy allowed identification of eosinophils in blood but not in adipose tissue although being detected by morphological analysis at low frequencies in some animals. A population not expressing CD45 and with the CH138A+ CD11b−MHC-II− phenotype, was found abundant and present at higher proportions in mesenteric than subcutaneous adipose tissue. The work reported here may be useful for further studies addressing the function of the described cells.