<p><i>Botrytis cinerea</i> can cause gray mold in more than 200 kinds of fruits and vegetables. Its rapidly developed resistance to pyrimethanil has become a key problem that restrict the application of pyrimethanil. At present, the research on the resistance mechanism of pyrimethanil remains insufficiently explored. In this study, we compared the mutation and expression level of the <i>CGS</i>, the target gene of pyrimethanil suggested by Fungicide Resistance Action Committee (FRAC), and the mitochondrial gene mutation related to pyrimethanil resistance. The results showed that the pyrimethanil resistance has nothing to do with the mutation and expression of the <i>CGS</i> gene of <i>B. cinerea</i>, but it is related to the E407K point mutation of mitochondrial gene <i>Bcmdl1</i>. Combined transcriptome analysis and exogenous amino acid reversal tests revealed that pyrimethanil resistance is associated with non-target enzyme genes, including 8 key genes involved in methionine synthesis (from serine/homoserine to S-adenosylmethionine) and 21 candidates linked to metabolic resistance. Significant changes in expression of several genes were observed in sensitive strains. Through the measurement of enzyme activities, the following enzymes were preliminarily associated with pyrimethanil resistance in <i>B.cinerea</i>: O-acetylserine hydrogenolysis synthase (OAS), methionine synthase (MS) (involved in methionine biosynthesis), and metabolic detoxification enzymes P450s and GSTs. The research outcomes are intended to clarify the multi-level resistance mechanisms of <i>B. cinerea</i> to pyrimethanil. This will offer a theoretical basis for effectively preventing or delaying fungicide resistance and implementing resistance management strategies.</p>