Viral Hemorrhagic Septicemia Virus (VHSV), is a World Organization for Animal Health (OIE) reportable fish pathogen found across the northern hemisphere, and lineage VHSV-IVb was first detected in the Great Lakes basin following sizable fish kills in the mid-2000’s. The biological and environmental factors that promote VHSV persistence following establishment are not well understood. Therefore, this study is investigating annual, spatial, and host species patterns of viral genetic structure in the upper St. Lawrence River (USLR), an ecosystem where VHSV has persisted for 20 years. The USLR supports large populations of invasive round gobies, and these fish are critical to provide an environment suitable to rapid viral reproduction, evolution of new viral isolates, and dissemination of those isolates to the native community.