Abstract Gray leaf spot, caused by the fungus Cercospora zeae-maydis, is an important foliar disease of corn (Zea mays) in the United States (U.S.). Application of foliar products that contain quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides is one way that corn farmers in the U.S. manage this disease. Since DMI and QoI fungicides pose medium to high risks of selecting for resistant fungal pathogens, it is important that fungicides from different classes be registered for use on corn to manage gray leaf spot. The fungicide pydiflumetofen (adepidyn; Syngenta Crop Protection, Greensboro, NC) is a new succinate dehydrogenase inhibitor (SDHI) that currently is being registered for use on corn in the U.S. The objective of this study was to establish the baseline sensitivity of C. zeae-maydis isolates collected from different areas of the U.S. to pydiflumetofen using in vitro assays. Effective concentrations of pydiflumetofen in which C. zeae-maydis isolates were inhibited by 50% (EC50 values) ranged from 0.0008 to 0.0101 μg/ml, with mean and median values of 0.0040 and 0.0038 μg/ml, respectively. These results can be used as part of a fungicide resistance monitoring program for C. zeae-maydis in the U.S.