Agropyron mosaic virus (AgMV), first described in Ontario, Canada, in 1937, has since been detected in Wales, Colorado, and Kansas, USA. AgMV is a positive-sense RNA virus in the genus Rymovirus, transmitted by the cereal rust mite Abacarus hystrix. In an Ohio survey in March to April of 2016, plants with virus-like symptoms, including stunting, chlorosis, and mosaic were collected from both planted soft red winter wheat fields and nearby volunteer wheat at 40 locations across 20 counties. Results of RNA sequencing and reverse-transcription (RT)-PCR assay confirmed the presence of AgMV in wheat samples with mosaic symptoms from one location each in Huron, Wayne, and Putnam counties, all of which were associated with nearby volunteer fields. Further testing by RT-PCR detected AgMV in only volunteer wheat in Wayne and Putnam counties and only production wheat in Huron county. AgMV was isolated from Huron and Wayne county samples, which were collected ∼60 miles apart, by rub-inoculation to the soft red winter wheat cultivar Freedom and then subsequent passaging three times. Infectivity test results agree with a previous report that AgMV infects wheat and rye but not oats. This is thought to be the first detection of AgMV in Ohio and in the Midwest United States.