Sex identification in teleosts with limited external sexual dimorphism remains a major challenge for aquaculture and reproductive management. To address this limitation, Pimelodus maculatus was used as a model to evaluate the potential of circulating sex steroids, including 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT), and the hormonal ratios T/E2 and 11-KT/E2 as minimally invasive biomarkers for sex identification. Females and males maintained in a recirculating aquaculture system (RAS) were evaluated at two sampling time points, in July and December 2024. Individual hormones showed limited or variable discriminatory performance between sampling time points, whereas the T/E2 ratio yielded the highest numerical classification metrics, with substantial to almost perfect agreement with ultrasonographic sex determination. The 11-KT/E2 ratio also showed discriminatory potential but exhibited greater variation between sampling time points. Histological analysis of a complementary group showed gonadal characteristics consistent with sexually mature adults that had not reached advanced stages of gonadal development, providing physiological context for interpreting the endocrine profiles under the experimental conditions. Overall, these findings demonstrate the potential of circulating endocrine indicators for minimally invasive sex identification in P. maculatus and support further investigation of the T/E2 ratio as a candidate composite endocrine indicator.
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