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The aim of the study was to analyze the amniotic fluid of chicken embryos for the concentration of steroid hormones, including testosterone, estradiol, and dehydroepiandrosterone (DHEA), and to determine whether these parameters differ between chicken breeds with different production rates. Eggs from various chicken breeds with different production rates were used for the study: Russian Snow White (egg), Pushkinskaya (meat and egg), Uzbek Fighting (sports), and the final Ross300 hybrid (meat). Starting on the eighth day of incubation, the amniotic fluid was analyzed for steroid hormone levels. An enzyme-linked immunosorbent assay revealed that the amniotic fluid of chicken embryos starting on the eighth day of incubation contained the steroid hormones testosterone, estradiol, and DHEA. From days 8 to 11, the levels of these hormones change only slightly, apparently reflecting the initial levels in the egg (maternal hormones). On day 12, a sharp increase in the concentration of the hormones studied occurs in hens of all breeds. The peak increase in hormone concentrations was recorded on days 13–15 of incubation. Differences in estradiol levels by breed during the embryonic period were generally minor. However, the highest concentrations of testosterone and DHEA in the amniotic fluid were recorded in the Ross300 broiler embryo group. Statistical analysis revealed a reliable positive correlation between the amniotic fluid testosterone-DHEA indices 0.83 (P≤0.01) in the group of Russian White breed embryos, 0.89 (P≤0.01) in the group of Pushkin breed embryos, as well as between the testosteroneestradiol indices in the group of Ross300 broiler embryos 0.72 (P≤0.01).