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Previous studies in Botrytis cinerea showed that resistance to methyl benzimidazole carbamates (MBCs) was mainly related to E198A/V/K and F200Y mutations of the β-tubulin gene, and E198V was the dominant mutation in the resistant subpopulation in Hubei Province of China, indicating that resistant mutations might influence fitness. However, little is known about the effect of each E198A/V/K mutation on fitness. In this study, the fitness and competitive ability of isolates with E198A/V/K mutations were investigated. Results showed that E198A/V/K isolates and wild-type isolates shared similar fitness components in terms of virulence, sporulation, conidial germination, oxidative sensitivity, and sclerotial production and viability. However, slower mycelial growth at 4°C, higher sensitivity to 4% NaCl, and increased sclerotial production percentage at 4°C were observed in the isolates with E198V, E198K, and E198A mutations, respectively. Competitive analysis showed that the wild-type subpopulation became dominant after three disease cycles in the absence of fungicide selection pressure, whereas the resistant subpopulation seized the space of the sensitive subpopulation upon MBC application. Unexpectedly, the frequency of E198V isolates decreased dramatically after the first disease cycle with or without fungicide selection pressure. These results suggest that MBC-resistant isolates suffer little fitness penalty but possess competitive disadvantages in the absence of fungicide selection pressure. Under fungicide selection pressure, E198V isolates could not compete with E198A/K isolates. According to the current results, there is a great possibility that the E198V mutation will lose dominance in the future in China.