Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum is an important leaf spot disease in the mid-Atlantic United States. Disease management approaches include use of resistant varieties, cultural control, and foliar fungicides. Frequent use of foliar fungicides can select for fungicide resistance within pathogen populations. Recently, the first report of quinone outside inhibitor (QoI) fungicide resistance in the United States was made based on a relatively small collection of P. nodorum isolates from Virginia. The objective of this study was to conduct a statewide, 2-year survey of P. nodorum populations in Virginia wheat and quantify frequencies of the target-site mutation that confers QoI resistance. A total of 318 isolates of P. nodorum were obtained from wheat collected at seven locations distributed throughout the wheat-growing regions of Virginia in 2018 and 2019. A previously designed pyrosequencing assay that detects the G143A substitution in the cytochrome b gene of P. nodorum was used to screen isolates for the presence or absence of the target site mutation. The G143A substitution was detected in all sampled fields. Among locations and years, frequencies of the mutation in P. nodorum populations ranged from 5 to 32% (mean = 19%). Thus, the QoI-resistance-conferring G143A mutation was widespread in P. nodorum populations in Virginia, and it occurred at a relatively high frequency. Results suggest that fungicides containing QoI active ingredients may not be effective for controlling SNB in Virginia and the surrounding region, and application of standalone QoI fungicides for disease control in wheat is not recommended.