Background: Sex determination and differentiation are complex processes shaped by a wide variety of molecular factors. In contrast to teleost species, many aspects of these processes remain poorly understood in basal non-teleost fishes such as the Siberian sturgeon (Acipenser baerii). Genetic sexing of this important aquaculture species now enables studies of undifferentiated males and females to identify factors involved in early sexual differentiation. Methods: Twelve undifferentiated Siberian sturgeon (six males, six females) were genetically sexed at 2.5 months of age. High-quality RNA was extracted from gonad samples, and transcriptomes were assembled using a reference dataset. Bioinformatic analyses were performed to identify sex-biased genes through differential expression analysis, Gene Ontology (GO) enriched terms, and classification of coding and non-coding sequences. Results: Genes potentially associated with sex differentiation were identified in gonadal tissue. Female-biased genes included classical estrogens production genes (hsd17b1, cyp19a1, foxl2) and immediate early response genes known to react rapidly to estrogens (jun-b, c-fos, egr1), as well as genes not previously linked to estradiol (di-ras2, ier2, aanat). The enriched Gene Ontology results suggested that melatonin signaling and hypothalamic pathways may also contribute to female differentiation. In males, the well-known factor tbx1 was upregulated along with novel candidates (plin1, nrxn3, chs2, mmp9). No sex-biased genes related to androgen production were identified. Conclusion By 2.5 months of age, sex-specific gonadal differences are already apparent in Siberian sturgeon. This study highlights the estrogen response pathway, including immediate early response genes described here for the first time in the context of fish gonadal differentiation. At the same time, an estrogen-independent ovarian pathway cannot be ruled out. Male differentiation appears to involve tbx1 together with new candidates for testis regulation in the absence of sex-biased androgen-producing enzymes. These novel genes expressed near the onset of sex differentiation merit further investigation.