Background: We aimed to investigate the relationship between sex hormone levels (estradiol (E2), total testosterone (TTest)) and anthropometric measurements, biochemical parameters, and body fat distribution of boys with obesity and gynecomastia, and the effects of all these variables, especially sex hormones, on fatty liver. Methods: This prospective study included 79 pubertal boys with obesity and gynecomastia between 10 and 18 years of age. The cases were divided into two groups as with (n = 48) or without (n = 31) fatty liver determined by ultrasonography (USG). Results: While E2 levels and age, body weight (BW), height, body mass index (BMI), waist circumference (WC), hip circumference, and fat mass of the patients were showing a statistically significant positive correlation, E2 levels had a negative correlation with high-density lipoprotein-cholesterol (HDL-C) (p<0.05). While age, BW, height, WC, and hip circumference of the patients were showing a statistically significant positive correlation with TTest, HDL-C levels, percent of body fat (PBF) (%) and percent of trunk fat (PTF) (%) had a statistically negative correlation with TTest (p<0.05). There was no significant difference in terms of sex hormone levels between the two groups with and without fatty liver (p>0.05). Conclusion: The main results of this study reveal that as the PBF and PTF increase, the TTest levels of the patients decrease and as the fat mass increases, the E2 levels increase significantly. The role of sex hormones in the pathogenesis of the fatty liver has not been clarified.