In this study, the levels of plasma estradiol-17β (E2) in farmed Anguilla japonica were measured to determine their sex. The analyses were performed for two different size groups (large group, Total length (TL): 61–69 cm; small group, TL: 53–60 cm). The anatomical and histological observations showed that the large group consisted of 29% males and 71% females; the small group, 54% males and 45% females. The gonad histology showed that in the large group, 88% of the eels had immature gonads with ongoing sexual differentiation, 12% were mature with completed sexual differentiation. In the small group, 87% of the eels had immature gonads. The plasma E2 hormone levels were higher in the females of both sizes. In the large group, the average plasma E2 in females was 415 pg/ml, which was significantly higher than the average of 109 pg/ml in males (P < 0.05). In the small group, the average plasma E2 hormone level was 618 pg/ml, which was much higher than the average of 108 pg/ml in males. Quantitative real-time PCR showed that zygote arrest 1 (zar 1) and zona pellucida glycoprotein 3 (zp3) were more highly expressed in females than male. In the H-E staining, an eel in the oil droplet containing ovary stage had a high level of plasma E2 (1500 pg/ml), while an eel with testis in the spermatocyte stage had a low (60 pg/ml). E2 is a potentially useful tool and could play an important role in sex determination in broodstocks. •Plasma estradiol-17β (E2) levels in farmed Anguilla japonica could indicate sex.•The plasma E2 hormone levels were higher in the females.•zar 1 and zp3 expression higher in females.•More reliable supply of broodstocks for artificial seedling production.