Glutathione S-transferases (GSTs) are detoxification enzymes important in cellular protection through their metabolism of endogenous and exogenous substrates such as chemotherapeutic agents, insecticides, herbicides, and byproducts of oxidative stress. All higher organisms express multiple GST isoenzymes with various substrate affinities, and rodent GSTs include members of the alpha, mu, pi, omega, theta, and zeta GST classes. A rho class GST (gstr) has been identified that is unique to fish and other aquatic organisms but has not been well characterized. In this study, we developed zebrafish (Danio rerio) gstr knockout lines and conducted toxicity studies using tert-butylhydroperoxide (tBHP) and cadmium (Cd) to test the role of gstr isoforms in protecting against contaminant-induced oxidative stress. Mutant zebrafish gstr knockout lines were developed through genome editing using CRISPR/cas9 targeting the coding sequence of zebrafish gstr. Wild-type and knockout zebrafish were exposed to tBHP and Cd at 96 hpf (hours post fertilization) and then analyzed for survival and developmental deformities. Whole embryo lipid peroxidation (LPO), glutathione concentrations (tGSH) and induction of two genes hsp70, a biomarker of cell stress, and gadd45bb, a biomarker of DNA damage, mRNA was measured to assess the effects of gstr knockout on sublethal embryonic oxidative damage. We found that gstr knockout zebrafish were more susceptible than wild-type embryos to the acute toxicity of 110 mg/L tBHP at 72 hours post exposure, and also that surviving gstr knockouts showed an increased prevalence of spinal curvature relative to wild-type fish. Exposure to a low dose of (55 g/L) of Cd caused a loss of whole embryo total glutathione (tGSH) concentrations and an induction in hsp70 mRNA relative to wild type zebrafish, consistent with sublethal oxidative stress. Transcriptional studies showed that gstr mRNA was refractory to induction by either tBHP or Cd. In conclusion, our studies indicate the rho class GST plays a functional role in protecting against chemical induced oxidative stress in zebrafish embryos. However, the lack of induction of gstr mRNA despite presence of AREs in the promoter region is consistent with a housekeeping role of the gstr gene, potentially in protecting against zebrafish embryos against oxidative damage. Supported by NIEHS Superfund ES04696.