The objective of this study was to assess trace element content in hair of Holstein dairy cows bred in the Leningrad Region of Russia and to calculate the site-specific reference intervals. Hair content of arsenic, boron, cadmium, cobalt, chromium, copper, iron, mercury, iodine, lithium, manganese, lead, selenium, silicon, tin, strontium, vanadium, and zinc in 148 cows during first (n = 50), second (n = 48), and third (n = 50) lactation periods of life was determined using inductively coupled plasma mass spectrometry. Dietary intake of trace elements corresponded to the adequate values according to national and international recommendations. Comparative analysis did not reveal any significant differences in hair content of main essential elements on the animals depending on the number of lactation. At the same time, the first-lactation cows had significantly (P < 0.05) lower concentration of lead in hair as compared to the third-lactation cows and a higher level of mercury as compared to the second-lactation cows. The reference intervals and 90% confidence intervals for the lower and upper limits were calculated in agreement with the American Society for Veterinary Clinical Pathology Quality Assurance and Laboratory Standard Guidelines. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.