This study investigated the effects of lactation period, foaling month and number, mare age, and regional factors on the quality parameters, amino acid composition, fatty acid profile, and nutritional indices of Kazakh mare’s milk under pasture conditions. A total of 240 milk samples were collected from Almaty and Zhambyl regions during the summer and autumn lactation periods. Standard physicochemical analyses determined fat, protein, casein, TS, and SNF contents, while amino acids were quantified via HPLC and fatty acids by GC. Significant seasonal differences were observed: summer milk contained higher PUFA (18.29%) and n-3 (5.71%) levels and exhibited lower SFA and AI values, indicating superior nutritional quality. Milk from younger mares (4 to 6 years) showed elevated essential amino acids and better lipid health indices compared to older mares. Zhambyl region samples had higher unsaturated fatty acids and SNF, while Almaty milk exhibited higher SFA and casein content. Amino acid profiling revealed that summer milk was enriched in glutamic acid, aspartic acid, serine, and histidine, whereas autumn milk contained more valine, leucine, methionine, and cysteine. PCA revealed distinct clustering based on season, mare age, and foaling period, confirming their substantial roles in shaping milk composition. These findings highlight that mare age, lactation period, and foaling timing significantly affect the nutritional quality of the mare’s milk. These results provide valuable insights for optimizing milk production and kumys fermentation strategies under traditional pasture-based systems.