Our purpose was to determine the effects on mammary gland development in prepubertal heifers given the anti-estrogen tamoxifen. Sixteen Holstein calves were randomly assigned to one of two treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were subcutaneously injected daily from 28 to 120 days of age with 0.3 mg/kg tamoxifen or carrier. At 120 days calves were euthanized and udders removed. Weight of trimmed parenchymal tissue (left rear quarter) was dramatically lower in TAM calves than in CON calves (p < 0.0003; 16.1 vs. 34.8 g). Parenchymal samples from three regions of the left rear quarter (lower, middle and outer regions) were processed for immunohistochemical staining for Estrogen Receptor α and Progesterone Receptor, myoepithelial cells, and label retaining cells. Overall, the proportion of neither ER nor PR labeled cells was impacted by TAM treatment. However, imaging analysis indicated a markedly higher intensity of ER expression in CON calves. TAM caused an increase in myoepithelial cell differentiation similar to what is seen in ovariectomy. We were able to effectively use a new technique of multispectral imaging to identify label retaining cells, which led to the discovery of an increase in the percentage of label retaining cells in TAM compared to CON. While treatment with the anti-estrogen tamoxifen reduced mammary parenchymal mass similarly to OVX, the mechanism(s) involved appear to differ. This suggests that the impacts of ovariectomy are only partially explained by the absence of estrogen.