Abstract: Monilinia fructicola is an important pathogen, causing brown rot in many fruit trees. Carbendazim is an effective fungicide for the control of this disease. In order to clarify the sensitivity of M. fructicola to carbendazim, and to understand the the genetic structure of this pathogen originated from different hosts, the sensitivities of 17 isolates from cherry, plum and peach were investigated in this study. The genetic diversity and genetic differentiation of 32 M. fructicola isolates from cherry and peach were also analyzed accroding to Tub2 nucleotide sequences. The results showed that the proportion of isolates with EC₅₀ < 1 μg/mL and > 1 μg/mL was 58.8% and 41.2%, respectively, among which the proportion of isolates with EC ₅₀ > 50 μg/mL was 17.6%. The population diversity of M. fructicola from peach was higher than that from cherry, with the nucleotide diversity of 3.25 × 10⁻³ and 9.04 × 10⁻³, respectively, and the haplotype diversity of 0.883 and 0.242, respectively. Significant genetic differentiation between the two populations was detected, and the degree of differentiation was medium with the F_ST value of 0.148. The haplotype network analysis showed that the populations from cherry and peach contained 3 and 9 haplotypes, respectively, within which 2 haplotypes were common. Different haplotypes evolved mainly in two ways. One pathway only showed one haplotype from peach population, and the other haplotypes formed the second pathway through different mutation steps. The host-specific test showed that the isolates from cherry had a certain host specificity, while the isolates from peach were less associated with the host. In conclusion, the resistant frequency of M. fructicola to carbendazim was high and the genetic structure of populations from different hosts was quite diverse.