Cucumber target leaf spot caused by Corynespora cassiicola has devastated greenhouse cucumber production. In our previous study, the monitoring of C. cassiicola resistance to carbendazim was carried out and a large number of resistant isolates which conferred various mutations (M163I&E198A, F167Y&E198A, F200S&E198A or E198A) in the β-tubulin of C. cassiicola were detected in the field. However, the single-point mutations M163I, F167Y, and F200S have remained undetected in the field. To investigate the resistance evolution mechanism of C. cassiicola to benzimidazoles, site-directed mutagenesis was used to construct alleles with single-point mutation M163I, F167Y, F200S, E198A, and double mutation M163I&E198A, F167Y&E198A or F200S&E198A in β-tubulin. Through PEG-mediated protoplast transformation, all the mutants except for the M163I mutation were obtained and conferred resistance to benzimidazoles. A similar sensitivity to benzimidazoles was observed between the F167Y and F200S mutations. However, in comparison to the F167Y or F200S mutations, double mutations (M163I&E198A, F167Y&E198A, F200S&E198A) and single mutation E198A caused significantly decreased sensitivity to benzimidazoles. The sensitivity to benzimidazoles for the double mutations (M163I&E198A, F167Y&E198A, F200S&E198A) did not differ from the single mutation E198A. Based on biological characteristics, the findings showed that the mutants conferring the F167Y or F200S mutations suffered fitness penalty, but not the mutants conferring the double mutations (M163I&E198A, F167Y&E198A, F200S&E198A) or the single mutation E198A. Thus, we concluded that to avoid the intrinsic fitness penalty and survive in the field, the double mutations (F167Y&E198A, F200S&E198A) evolved from the single mutations F167Y and F200S, respectively, and not the single mutation E198A.