Barley yellow dwarf virus (BYDV), an economically important virus, infects small grain cereal crops and over 150 other Poaceae species. BYDV infection plays an important role in competition among grasses in non-managed systems, but many grasses remain unexamined as potential BYDV hosts. This study examined grass species that have not been reported as BYDV hosts but are commonly encountered in non-managed grasslands throughout the United States and Canada. Laboratory inoculations with BYDV-PAV using the aphid vector Rhopalosiphum padi were performed to examine the ability of 13 grass species and barley to be infected with the virus; eight of the grass species were not documented previously as virus hosts. Serological and molecular assays were used to confirm BYDV-PAV infection. Plant height, number of leaves, number of tillers and weight were recorded to evaluate susceptibility or sensitivity to BYDV. Infection with BYDV was experimentally achieved for the first time on Achnatherum lettermanii, Achnatherum occidentale, Achnatherum thurberianum, Danthonia intermedia, Poa fendleriana, Sporobolus airoides and Sporobolus cryptandrus, but not on Alopecurus pratensis and Elymus wawawaiensis. Infection was confirmed in Bromus inermis, Elymus elymoides, Poa bulbosa, Poa secunda and Hordeum vulgare, which served as controls. BYDV infection caused reductions in plant height on P. bulbosa and P. fendleriana. BYDV-infected P. secunda had more leaves per plant compared to healthy plants of the same species. BYDV-infected A. lettermanii exhibited reduced dry weight in both below-ground and above-ground tissue. These findings have implications for the management and conservation of grassland habitats.