A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the muskellunge (Esox masquinongy) , against VHSV‐IVb challenge. Seven weeks (539 degree‐days) following vaccination with 10 μg of either pVHSivb‐G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque‐forming units (pfu)/mL of VHSV‐IVb. Fish vaccinated with pVHSivb‐G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree‐days) postchallenge (11 weeks postvaccination) in 100% of muskellunge vaccinated with pVHSivb‐G compared with only 12% of plasmid‐control‐vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb‐G–vaccinated rainbow trout Oncorhynchus mykiss challenged 7 d (100 degree‐days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV‐IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species.