As a lactation source, donkey milk contains a higher percentage of n-3 polyunsaturated fatty acids in the milk fatty acid profile than cow's milk, especially the percentage of α-linolenic acid (ALA). We hypothesized that the ratio of dietary concentrate/roughage could influence the composition of the fatty acid (FA) profile of donkey milk, and that the substitution of low-quality roughage with high-quality roughage could improve the composition of the polyunsaturated fatty acid profile of donkey milk. The purpose of this experiment was to investigate the effects of the partial dietary replacement of low-quality roughage with alfalfa hay to increase the proportion of roughage in the diet on the FA profiles in the milk of lactating donkeys, with the goal of optimizing the FA profiles of donkey milk. The trial design was a single-factor randomized design. Sixteen Dezhou donkeys of similar age, weight, parity, and lactation days were selected and equally divided (