Bovine mammary development begins before puberty with the elongation and branching of ducts and stromal remodeling. This process is highly regulated, and in mice impacted by the presence of innate immune cells which cluster near developing ductal structures. For the first time, we studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in prepubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the 3 cell types in formalin fixed, paraffin embedded mammary tissue from prepubertal female calves in archived sets. The first set (ONT) included samples (n = 4 per week) from birth to 6 wk of age. A second set (OVX) allowed for examination of the potential influence of ovaries on innate immune cells. It included samples from 19 animals who were either intact or ovariectomized 30 d before sample collection. These animals were 90, 120 or 150 d old at examination. The third set (EST) allowed for examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. This set included samples from 10 calves given estrogen implants (n = 6) or placebo (n = 4) at 56 d of age. Calves were sampled at 70 d of age. Eosinophils were identified via LUNA staining, mast cells by May–Grunewald Giemsa staining, and macrophages with immunoflourescence. Stroma was examined in 20 images from each animal of the NEAR (<100 μm) and FAR (>100 μm) locations relative to the epithelial ducts. Results were expressed as cells/mm². More eosinophils and mast cells were observed in NEAR compared with FAR stroma in the ONT (P < 0.001) and OVX (P < 0.05) but not EST. More macrophages were also observed in NEAR compared with FAR in ONT (P < 0.001). Treatment (age, ovariectomy or estrogen) had no impact on stromal eosinophils. Age tended to affect mast cell numbers in ONT, with the most mast cells observed at 14 d of age. In summary, we show for the first time that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure and physiological state of development. We suggest there may be a role for these cells in control of bovine mammary development.