The study was carried out to determine the effect of 2 ecological regions and lactation period on chemical (P < 0.01) and amino acid (P < 0.01) composition and nitrogen fraction (P < 0.01) of mares grazing on pastures at 1700 m (vegetation is dominated by shrubs and sub-shrubs) and 2200 m (vegetation is dominated by grasses, forbs, sub-shrubs) above sea level. The animals were kept under extensive pasture conditions and received no additional feed supplements. Milk samples were collected monthly from May to July and from May to August from mares grazing at 1700 m (Mountains) and 2200 m (Highlands), respectively. Total solids (11.39–10.47% and 11.56–10.86%), milk fat (1.80–1.33% and 1.83–1.60%) and ash (0.48–0.26% and 0.55–0.30%) content of milk for the Mountain and Highland regions respectively were decreased to the end of lactation period. The average percentages of casein to whey protein ratio were 52: 47 and 50:42 for milk obtained from Mountain and Highland pastures. The highest content of essential amino acids were obtained in May milk for both regions (1251 and 1284 mg/100 g of milk) and the lowest content were in July (852 mg/100 g of milk) and August milk (880 mg/100 g of milk), but the percentages of essential amino acids in protein of mare’s milk were the highest in June (49%) and July milk (49%) produced at 1700 m and 2200 m respectively. The results of the study have shown that the changes in chemical composition, nitrogen fraction, and amino acid profile of the mare’s milk during lactation period are influenced by geographical location of pastures.