The frequency of arthropod-borne viral disease in naïve hosts is subject to change based on complex interactions among vector, host, virus, and external factors (e.g., climate). Thus, continual monitoring for both disease susceptibility and host infection dynamics is needed, especially for viruses that have proven detrimental to the health of wildlife hosts of conservation concern. The Greater Sage-grouse (Centrocercus urophasianus) is a gamebird of ecological and economic importance in the western United States for which population declines have led to a Near Threatened conservation status. Although these declines have mainly been attributed to habitat loss, West Nile virus (WNV) also poses a threat, with regional transmission potentially fueled by anthropogenic landscape alterations that may facilitate mosquito breeding. With limited WNV monitoring in Greater Sage-grouse after recognition of high susceptibility to mortality early after its initial detection in the western United States, the potential long-term impacts of WNV on this species are poorly understood. We used the plaque reduction neutralization test of filter paper strip–eluted sera to assess for anti-WNV antibodies, indicating prior infection, in opportunistically collected samples. From 2020 to 2022, 85 Greater Sage-grouse in Wyoming were sampled; none had anti-WNV antibodies. This result corroborates findings of previous studies documenting low seroprevalence. With the tenuous conservation status of the species, all potential population health risks should be considered in future management strategies, especially in the face of changing climate and landscapes.