Ass’s milk yield and chemical composition were investigated in two lactations at d 28, 45, 60, 75, 90, 105, 135 and 150 after parturition. Milk yield per milking (averaging 740 mL) of 6 asses of Martina Franca (n = 3) and Ragusana (n = 3) breeds, which quickly adapted to the milking machine procedures, increased in the second (or third) milking per day but was unaffected by stage of lactation or breed. Year of lactation, stage of lactation, breed and milking time did not influence the gross composition of the milk, except for fat and protein contents. The overall average protein percentage (1.72 g·100 g–1 of milk), showing a significant negative trend throughout lactation, was characterised by low casein (47.3% of crude protein) and whey protein contents (36.9% of crude protein). A specific whey protein profile was also found, being the relative percentage on total whey protein 4.48%, 6.18%, 29.85%, 21.03% and 22.56% for respectively lactoferrin, serum albumin, β-lactoglobulin, lysozyme and α-lactoalbumin. The low average fat content of ass’s milk (0.38 g·100 g–1 of milk), showing a high individual variability, was significantly affected by the year of the study; the lipid fraction was also characterised by high levels of linoleic (average 8.15 g·100 g–1 of total fatty acids) and linolenic acid (average 6.32 g·100 g–1 of total fatty acids). The ash content of ass’s milk (0.39 g·100 g–1 of milk) was constant throughout the experimental period and showed high levels of Ca and P, the ratio of which, ranging between 0.93 and 2.37, was on average 1.48.