Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.