Barley yellow dwarf (BYD), caused by ten different species of viruses from the Luteoviridae family, is an economically important viral disease of small grains in the US Northern Great Plains. Identification of virus species in the region associated with BYD is critical to deployment of specific effective resistance genes. However, a comprehensive survey of virus species using modern diagnostic tools has not been done before in the US Northern Great Plains. In this study, species composition of viruses associated with BYD in small grains of Minnesota and South Dakota were determined by sampling BYD infected tissue from a large geographical area over three years. Whole genomic RNA was extracted from leaf tissue and reverse transcription polymerase chain reaction (RT-PCR) was conducted to detect infection of BYD and classify into species using primers from a two-step multiplex PCR protocol. Out of 463 samples collected from Minnesota and South Dakota from 2013 to 2015, about 50% (n = 222) of the samples tested positive for BYD. The majority of virus infected samples (n = 146) were identified as the Barley yellow dwarf virus PAV. These results indicate that the RT-PCR protocol used in this study is a viable molecular diagnostic tool for detection of BYD causing viruses in field collected samples. Moreover, the results indicate that Barley yellow dwarf virus PAV is most the dominant species associated with BYD in the US Northern Great Plains. Due to the species-specific nature of resistance to BYD these results can also guide breeding strategies in small grains for BYD management.