Very little is known about the udder characteristics, partitioning of milk in the mammary gland and efficiency of machine milking in donkeys. This study aimed to evaluate the characteristics of the udder and teats, milk yield in relation to pulsation rates (90, 120 and 150 cycles/min), milk partitioning in the mammary gland, composition of the spontaneously removed and residual milk fractions and milking efficiency. Forty-one healthy Martina Franca jennies in the third month of lactation and routinely milked twice daily were used in three studies. Udder characteristics were evaluated by direct measurements and ultrasonographic scanning. Residual milk was obtained by milking after an oxytocin administration (40 IU i.m.). The prevalent shapes were ‘bowl’ for udders and ‘conical’ for teats. After milking the udder characteristics decreased within a range from −11·6% (udder depth) to −25·7% (diameter of teat at the base). The internal structures of the udder resulted as several pockets of ducts empting directly into the teat. The pulsation rate of 120 cycles/min improved (P<0·05) the milk yield in comparison to the 90 and 150 cycles/min, reduced the residual milk fraction, thus improved (P<0·05) milking efficiency. Residual milk composition had higher (P<0·05) fat content and somatic cell count than the spontaneously removed milk fraction. The udders revealed several pockets of ducts empting into the teat instead of a single cisternal cavity and showed a certain compliance. The use of 120 cycles/min pulsation rate improved milking efficiency.