This study aimed to examine the teat characteristics in relation to the animal temperament during milking in the Anatolian buffaloes using ultrasonographic, histomorphological and immunohistochemical methods. The teat canal length (TCL), teat wall thickness (TWT), teat cisternal diameter (TCD), teat diameter (TD), teat length (TL), and teat circumference (TC) values in docile (n = 5) and nervous (n = 7) buffaloes were measured at the 0th, 3rd and 6th minute of stimulation. In additional experiments, comparative histomorphology and immunohistochemical examinations of buffalo (n = 7) and cow teats (n = 8) were performed. It was determined that post-stimulation mean TCL values were significantly higher in nervous buffaloes than those of teats in docile buffaloes (p < .05). A significant positive correlation between TCD and TD, TL and TC in both docile and nervous buffaloes was noted (p < .05). Unlike nervous buffaloes where only 3/14 teat canals were open by 3rd minute of milking stimulation, almost all (9/10) teat canals were observed opened in docile buffaloes. There were fewer muscle but more collagen bundles in buffalo teats compared with cow teats. It seems that temperament of animal during milking effects the milking efficiency, and in nervous buffaloes, probably the stimulation alone may not be sufficient for opening of the teat canal and hence achieve complete milking.