The necrotrophic pathogen Sclerotinia sclerotiorum has a global distribution and a wide host range, making it one of the most damaging and economically important of all plant pathogens. The current study found that fluxapyroxad, a typical succinate dehydrogenase inhibitor fungicide, had a strong inhibitory effect against S. sclerotiorum, with mean effective concentration for 50% inhibition (EC50) values ranging from 0.021 to 0.095 µg/ml. Further investigation of five highly resistant S. sclerotiorum mutants, with EC50 values of 12.37 to 31.36 µg/ml, found that fluxapyroxad resistance was accompanied by a certain cost to fitness. All of the mutants were found to have significantly (P < 0.05) reduced mycelial growth and altered sclerotia production in artificial culture, as well as reduced pathogenicity, compared with wild-type isolates, with one mutant completely losing the capacity to infect detached soybean leaves. Sequence analysis demonstrated that four of the mutants had point mutations leading to amino acid changes in the SsSdhB subunit of the fungicide target protein succinate dehydrogenase. In addition, two of the mutants were also found to have amino acid changes in the predicted sequence of their SsSdhD subunit, while the fifth mutant had no changes in any of its SsSdh sequences, indicating that an alternative mechanism might be responsible for the observed resistance in this mutant. No cross-resistance was found between fluxapyroxad and any of the other fungicides tested, including tebuconazole, prochloraz, dimethachlone, carbendazim, procymidone, pyraclostrobin, boscalid, fluazinam, fludioxonil, and cyprodinil, which indicates that fluxapyroxad has great potential as an alternative method of control for the Sclerotinia stem rot caused by S. sclerotiorum, and which could provide ongoing protection to the soybean fields of China.