Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 α (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that (1) HNF4A identifies putative buffalo mammary stem/progenitor cells and (2) the number of mammary stem/progenitor cells increases during mastitis. Thirteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-μm-thick sections. Based on histomorphology of mammary glands, physiological stages were estimated to be nonlactating (n = 4 animals) and mastitis (n = 9 animals). In total, ~22,000 cells were counted (10 microscopic fields/animal; n = 13 animals), of which 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in nonlactating animals was higher compared with that in mastitic animals (47.3 vs. 37.3%), which was likely due to loss of MEC caused by infection. HNF4A staining was observed in nuclei of MEC of ducts, alveoli and stromal cells. Basal location and low frequency of basally located HNF4A positive cells (ranges from 0.4 to 4.5%) was consistent with characteristics of mammary stem cells. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher during nonlactating stage than mastitis (8.73 ± 1.71 vs. 4.29 ± 1.19%; P = 0.07). MEC proliferation (assessed by immunohistochemical expression of Ki67) was higher in mastitic glands in comparison to nonlactating glands (15.3 ± 5.7% vs. 0.53 ± 0.1%; P = 0.03). This is the first report outlining the expression of HNF4A as a putative mammary stem/progenitor cells of buffalo mammary gland; and evaluation of MEC proliferation in naturally infected mastitic buffaloes.