is a destructive fungal pathogen that causes major crop losses worldwide. The fungicide pencycuron is commonly used to control Rhizoctonia diseases, but some anastomosis groups (AGs) such as AG-7 are generally resistant to pencycuron. This study discovered a sensitive AG-7 isolate (No.214), and compared its genomics and transcriptomics to a resistant isolate (No.213). PacBio whole-genome sequencing and Illumina RNA sequencing (RNA-seq) revealed that No.214 has a whole-genome duplicated and pentaploid genome, showed broad transcriptional changes but lacked key detoxification genes in response to pencycuron. In contrast, No. 213 has a diploid genome and the transcriptional responses were mostly xenobiotic detoxification genes such as cytochrome P450 (). Functional validation in confirmed conferred pencycuron resistance. Collectively, this work advances the understanding of cytochrome P450-mediated pencycuron resistance and suggests genomic complexity of AG-7 isolates may influence pencycuron susceptibility.