Perilla frutescens is a traditional medicinal plant and functional food in Asian communities, characterized by distinct red and green leaf types that have significant phenotypic and medicinal implications. However, the genetic mechanisms controlling anthocyanin synthesis in this species remain unclear. Genetic analysis serves as a powerful tool for investigating the pivotal genes and regulatory mechanisms governing anthocyanin accumulation in red and green perilla. In this study, an F2 segregation population was constructed from a hybrid of red and green perilla, and representative samples were subjected to mix-sequencing using BSA-seq and BSR-seq. A 6.0 Mb candidate region on chromosome 8 was identified, pinpointing PfMYB113b, PfC4H1, and PfF3H as key genes involved in anthocyanin biosynthesis. The insertion of a repeat sequence in the promoter of PfMYB113b leads to alterations in gene expression levels. Furthermore, PfMYB113b regulates the transcription of PfC4H1 and PfF3H, thereby influencing anthocyanin synthesis. These findings enhance our understanding of the genetic regulatory mechanisms underlying leaf coloration in perilla.