BACKGROUND Botrytis cinerea, the causal agent of gray mold, is one of the top 10 fungal pathogens in the world. Propamidine, an aromatic diamidine compound, exhibited both protective and therapeutic effects against B. cinerea. However, the resistance risk and mechanism of B. cinerea to propamidine are unclear. RESULTS Twelve high and stable resistant mutants were obtained from B. cinerea B05.10 by fungicide induction. Compared with the parental strain, the biological fitness of the mutants, including growth rate, spore germination, pathogenicity, and oxalic acid decreased significantly. There was no cross‐resistance among propamidine and other commonly used fungicides, while the efficacy of propamidine against the resistance mutants declined. In addition, the cell membrane permeability, substance metabolism, and defense enzyme activities of the resistant mutants were significantly increased compared with the wild strain. Whole‐genome sequencing of all resistant mutants found that there were 32 SNPs and nine InDels. Importantly, nine common single‐point mutant genes in the exon region were found in all 12 resistant mutants, and these genes were related to multiple pathways in vivo, indicating that many factors contributed to the formation of propamidine resistance. CONCLUSION These data suggested the resistance risk of B. cinerea to propamidine was low to moderate and the mechanism of propamidine was different from that of the existing fungicides. These results will increase understanding of the resistance mechanism of propamidine and provide a critical basis for the rational design of pesticide molecules based on targets. © 2022 Society of Chemical Industry.