The population of Botrytis cinerea in strawberry-producing areas in China shows increasing resistance to the fungicide pyraclostrobin. A survey with 206 isolates collected between 2024 and 2025 in Xinxiang confirmed resistance in 100% of the samples. The average resistance level increased over the period. The EC50 values rose from 75.31 μg/mL to 85.08 μg/mL. The number of isolates with high resistance also grew. Molecular analysis identified the G143A mutation in the BcCytb gene as the main mechanism. The gene lost an intron. The event caused a substitution of glycine with alanine at the action site. The change reduced the binding affinity of the fungicide to the mitochondrial target. Molecular modeling confirmed the drop in binding energy. The resistant isolates also showed an increase in the expression of the BcCytb gene. The expression was up to 8.5 times the levels observed in sensitive isolates. The increase occurred even without exposure to the fungicide. This pattern ...