Frogeye leaf spot (FLS) is a foliar disease of soybean (Glycine max (L.) Merr.) caused by Cercospora sojina Hara. Application of fungicide products that contain quinone outside inhibitor (QoI) active ingredients has been one of the major tools used in the management of this disease, but since 2010, QoI-resistant C. sojina isolates have been confirmed in over 20 states in the United States, including Indiana. In the summers of 2019 and 2020, 406 isolates of C. sojina were collected from 32 counties across Indiana and screened for QoI resistance using a PCR-RFLP method. An In vitro fungicide sensitivity test was also performed on a subset of isolates to evaluate their sensitivity to three QoI fungicides: azoxystrobin, pyraclostrobin, and picoxystrobin. A discriminatory dose of picoxystrobin was established as 1 μg/ml by testing five concentrations (0.001, 0.01, 0.1, 1, and 10 µg/ml). QoI-resistant isolates were found in 29 counties, and 251 out of the 406 isolates (61.8%) were confirmed to be resistant to QoI fungicides based on PCR-RFLP results. Partial nucleotide sequences of the cyt b gene from four resistant and four sensitive isolates corroborated the presence and absence of the G143A mutation, respectively. Results from the sensitivity assays with discriminatory doses of azoxystrobin (1 μg/ml) and pyraclostrobin (0.1 μg/ml) also supported the findings from the PCR-RFLP assay, as all QoI-resistant isolates were inhibited less than 50% relative to a no-fungicide control when exposed to these doses. Resistant isolates harboring the G143A mutation also exhibited resistance to picoxystrobin. The effective concentrations to inhibit mycelial growth by 50% relative to the non-amended control (EC50) in QoI-sensitive isolates ranged from 0.087 to 0.243 μg/ml with an overall mean of 0.152 μg/ml while EC50 values in QoI-resistant isolates were established as >10 μg/ml for picoxystrobin. Results from this study indicated that QoI-resistant C. sojina isolates are spread throughout Indiana and exhibit cross-resistance to QoI fungicides.