Glutathione S-transferases (GST) are considered among the most controversial biomarkers of water pollutants in fish with little known about factors influencing their activities. The objective of this study was to investigate how gender, dose, ploidy, and sampling time alter hepatic GST activities in African catfish (Clarias gariepinus) following β-naphthoflavone (β-NF) injection. Newly matured male and female diploid and triploid fish were intraperitoneally (i.p.) injected with 0, 15, or 75 mg/kg of β-NF, and livers were excised 24, 48, and 72 h post-injection. Results showed that hepatic GST activities were significantly inhibited by both doses of β-NF. Inhibition was greater in females than males, but no significant differences were observed between diploid and triploid fish. Enzymatic activities differed over time with lowest levels 72 h post-injection. These results extend our understanding of GST activity in fish and highlight the necessity of considering confounding factors when comparing different studies.