Hormonal sex reversal can produce monosex fish stocks and provide insights into their gamity and reproductive physiology. However, paradoxical effects have been reported in several fish species that remain largely ignored as anomalies, particularly those of masculinisation. As a first step, this study examined reproductive viability of paradoxically masculinised Gambusia holbrooki produced following oral administration (20–100 mg/kg feed) of a feminizing hormone diethylstilbestrol (DES). Contrary to expectation, all treatment groups produced 100% male populations. Survival, mating behaviour, gamete production, breeding output as well as expression of anti-Mullerian hormone (amh), ovarian (cyp19a1a) and brain (cyp19a1b) aromatase of masculinised fish were also examined. Survival (≤ 54.1 ± 7.3%) at termination of DES treatment was significantly lower compared with controls (88.6 ± 4.3%) but remained unaffected post treatment. Gonopodium thrusting frequency (33 ± 9.8 per 10 min) was not significantly different to untreated males just as sperm abundance (3.9 ± 1.5 × 10⁸/male) and their motility (88.6 ± 29.1%). Importantly, paradoxically masculinised fish mated with virgin females and produced clutch sizes (22 ± 4) and progeny survival (87.0 ± %) that were comparable to that of untreated males. Masculinised testes showed high amh and low cyp19a1a expression, a pattern resembling those of untreated males. Production of paradoxically sex-reversed males with a capability to produce viable offspring has not been reported previously in this or other fish species. The outcomes support a feed-back regulation of oestrogenic pathways in this viviparous fish and could be useful for ecological applications such as controlling invasive fish populations.