Mutations in the succinate dehydrogenase (sdh) complex subunit B are associated with different levels of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides in Botrytis cinerea, the causal agent of Botrytis fruit rot (BFR) of strawberry. Due to the emergence of B. cinerea populations resistant to the various SDHIs deployed in strawberry fields, it is important to elucidate the genotype of such populations to limit the use of at-risk SDHIs. Thus, the objective of this study was to develop and validate a High-Resolution Melting (HRM) assay for the rapid detection of sdhB mutations using conidia from infected strawberry tissue. Two sets of HRM markers were designed based on the sdhB gene of B. cinerea from strawberry to detect either the H272R/Y or the P225F mutations. SdhB characterization and mutation detection of 25 B. cinerea isolates collected during the 2017-18 Florida strawberry season were performed using the HRM and Sanger sequencing assays. Both assays confirmed the presence of the H272R and H272Y mutations in four and two isolates, respectively, whereas the P225F was only detected in one isolate. Our study presents a fast and cost-effective method to detect frequent (H272R/Y) and powerful (P225F) sdhB mutations. Furthermore, HRM analysis could be implemented in plant diagnostic clinics to monitor predominant genotypes in strawberry fields and inform growers about SDHI fungicides with high risk for selection of resistance that would limit BFR control.