Anthocyanin biosynthesis and accumulation determines the colour of tuber flesh in potato (Solanum tuberosum) and influences nutritional quality. However, the regulatory mechanism behind anthocyanin biosynthesis in potato tuber flesh remains unclear. In this study, we identified the Pigmented tuber flesh (Pf) locus through a genome-wide association study using 135 diploid potato landraces. Genome editing of two tandem R2R3 MYB transcription factor genes, StMYB200 and StMYB210, within the Pf locus demonstrated that both genes are involved in anthocyanin biosynthesis in tuber flesh. Molecular and biochemical assays revealed that StMYB200 promotes StMYB210 transcription by directly binding to a 1.7-kb insertion present in the StMYB210 promoter, while StMYB210 also regulates its own expression. Furthermore, StMYB200 and StMYB210 both activated the expression of the basic helix-loop-helix transcription factor gene StbHLH1 and interacted with StbHLH1 to regulate anthocyanin biosynthesis. An analysis of the StMYB210 promoter in different diploid potato accessions showed that the 1.7-kb insertion is associated with flesh colour in potato. These findings reveal the genetic and molecular mechanism by which the Pf locus regulates anthocyanin accumulation in tuber flesh and provide an important reference for breeding new potato varieties with colourful flesh.