Anthracnose fruit rot disease, caused by Colletotrichum spp., is the most significant disease problem of commercial strawberry (Fragaria × ananassa) production in the southeastern United States. The hot, humid weather and continuous rainfall in Georgia make Colletotrichum-induced fruit rot a widespread problem in strawberry production. In order to control this disease, growers mainly rely on preventive fungicide applications from flower bud emergence to harvest. The most commonly used single-site fungicides are quinone outside inhibitors (QoIs); the QoI active ingredients azoxystrobin and pyraclostrobin are utilized to manage anthracnose fruit rot. In 2019, we collected 108 strawberry fruits with visible rot symptoms from seven different strawberry farms in Georgia. These farms had received multiple applications of QoI fungicides during the 2019 growing season, as well as in previous seasons. Sensitivities to pyraclostrobin were assessed on 1% malt extract agar using a mycelial growth inhibition assay. Our results demonstrated that a majority of Colletotrichum isolates collected in 2019 were not inhibited by pyraclostrobin, suggesting a growing resistance issue with the QoI fungicides. A PCR-restriction fragment length polymorphism assay showed the presence of the G143A mutation in all QoI “resistant” C. acutatum isolates and none for isolates labeled “reduced sensitivity” or “sensitive”. These results further prove that C. acutatum isolates with the G143A mutation are highly resistant to the QoI fungicide. These findings suggest that there is a high risk of resistance development associated with using pyraclostrobin (likely all QoIs) for controlling anthracnose fruit rot of strawberry in Georgia.