The polar bear is an iconic Arctic species, threatened by anthropogenic impacts such as pollution and climate change. Successful reproduction of polar bears depends on a functioning steroid hormone system, which is susceptible to effects of persistent organic pollutants. The present study is the first study to report circulating concentrations of nine steroid hormones (i.e., estrogens, androgens and progestagens) in female polar bears (Ursus maritimus). The aim of the study was to investigate the effects of age, condition, location and reproductive status on steroid profile in female polar bears. Levels of pregnenolone (PRE), progesterone, androstenedione (AN), dehydroepiandrosterone (DHEA), testosterone, dihydrotestosterone, estrone (E1), 17α-estradiol (αE2) and 17β-estradiol (βE2) were quantified in blood (serum) of free-living female polar bears (n = 15) from Svalbard, Norway, by gas chromatography–tandem mass spectrometry (GC–MS/MS). Concentrations of androgens, estrogens and progestagens were in the range of 0.02–166, 0.01–1.49 and 0.16–17.1 nmol/L, respectively. A statistically significant negative correlation was found between E1 and DHEA, and a positive correlation between E1 and βE2. Additionally, negative relationships were found between body mass and cholesterol, contour body length and cholesterol, and head length and PRE, while a positive relationship was found between PRE and cholesterol. The steroid profile suggests that AN and the sex steroids are primarily synthesized through the Δ-4 pathway in polar bears, similar to rodents. The large individual variability in steroid levels reported here most likely reflects the differences in reproductive status of the female polar bears during mating season. The steroid data establish reference values of steroid hormones and may be applied in further studies on polar bears endocrine system and anthropogenic threats to polar bear reproduction.