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The gross morphology of the mammary gland in buffalo is the foundation that is necessary for the process to improve the species for agricultural use. This study determined the mammary gland's anatomical, histological and imaging structure in the Romanian buffalo. The research material was represented by ten udders, five udders from adult buffaloes and five udders from young females slaughtered in the abattoir. The udders were collected immediately after slaughtering the animals, then transported to USAMV Cluj‐Napoca, Romania, examined macroscopically, histologically and through imagistic methods. The morphological examination tracked the topography, external conformation, vascularization and innervation of the mammary gland in the buffalo. The histological examination highlighted differences between the ductoacinary structures of the mammary gland parenchyma in adult buffaloes compared with young females. The imaging examination showed the detachment of the middle mammary artery and the arterial anastomoses established between the mammary arteries on the left side of the udder and those on the right side and between the arteries of the quarters on the same side. This research can be theoretical and practical support to other studies in establishing the most accurate diagnosis and therapeutic protocol in various clinical pathologies of the udder in buffalo. [ABSTRACT FROM AUTHOR]