Hair cortisol analysis is a helpful tool for non-invasive measurement of the long-term activity of the HPA (hypothalamic-pituitary-adrenal) axis. Long-term exposure to stressors affects, among others, animals staying in shelters for homeless animals. The study aimed to assess the hair cortisol level in cats from 8 different shelters. Hair grows on average about 1 cm per month. Therefore, it was concluded that the cortisol level in the hair closer than 1 cm to the skin reflected the cortisol level in cats during their stay in the shelter (A period). In contrast, the cortisol level in the rest of the hair corresponded to the cortisol level preceding the cat’s admission to the shelter (B period). Hair cortisol levels in the cats were higher before they arrived at the shelter than during their stay. Hair cortisol levels in the period before the shelter were higher in females than they were in males. On the other hand, during the stay at the shelter, the opposite was true: the cortisol level in females was lower than in males.