The objective of the present study was assessment of gender differences in hair trace element content in English Thoroughbred horses (North Caucasus, Russia) using ICP-DRC-MS and calculation of the reference values. Trace element content in mane hair of 190 stallions and 94 mares (3-7 years old) bred in North Caucasus (Russia) was assessed using inductively coupled plasma mass spectrometry. Mane hair Co, Cr, Mn, Li, Si, and Sr levels in mares exceeded those in stallions by 77%, 63%, 64%, 42%, 39%, and 64%, respectively. Hair Fe and Si content was nearly twofold higher in female horses as compared to the males. Only hair Zn content was 5% higher in stallions as compared to mares. In addition, mares were characterized by 63%, 65%, 29%, and 40% higher levels of As, Pb, Sn, and Ni levels in hair as compared to the respective values in stallions. In turn, hair Al and Hg were more than twofold higher in mares than in stallions. The reference intervals of mane hair content (μg/g) for Co (0.006-0.143), Cr (0.028-0.551), Cu (4.17-6.84), Fe (10.11-442.2), I (0.026-3.69), Mn (0.551-12.55), Se (0.108-0.714), Zn (97.43-167), Li (0.011-0.709), Ni (0.060-0.589), Si (0.665-29.12), V (0.006-0.584), Al (1.98-168.5), As (0.006-0.127), Cd (0.002-0.033), B (0.494-16.13), Pb (0.018-0.436), Sn (0.002-0.144), Sr (1.0-9.46), and Hg (0.0018-0.017) in the total cohort of horses were estimated using the American Society for Veterinary Clinical Pathology Quality Assurance and Laboratory Standard Guidelines. The reference intervals were also estimated for stallions and mares bred in North Caucasus (Russia) and may be used for interpretation of the results of hair trace element analysis in horses.