Endometrial cancer is known to be an estrogen-dependent cancer, and it is believed that exposure to estrogens increases the risk of developing endometrial cancer in the absence of progesterone. Estrogens and estrogen metabolites may help to predict the risk of endometrial hyperplasia (EH) with atypia. Estrogens and estrogen metabolites were measured by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in the first morning urine sample collected from 150 patients with EH and 50 healthy premenopausal women in the study. In healthy premenopausal women, the level of 16α-hydroxyestrone (16α-OHE1) in the overweight group was significantly higher than that in the lean group (p < 0.05). The levels of 4-hydroxyestradiol (4-OHE2) and 16α-OHE1 were elevated in the AEH group as compared to that in the control group (p < 0.05). Overweight is related to EH incidence as it causes an imbalance of estrogen metabolites. This study provides identifies potential biomarkers for estrogen-induced AEH.© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.