Sweet cherry is a diploid tree species and its fruit skin has rich colours from yellow to blush to dark red. The colour is closely related to anthocyanin biosynthesis and is mainly regulated at the transcriptional level by transcription factors that regulate the expression of multiple structural genes. However, the genetic and molecular bases of how these genes ultimately determine the fruit skin colour traits remain poorly understood. Here, our genetic and molecular evidences identified the R2 R3 MYB transcription factor Pav MYB10.1 that is involved in anthocyanin biosynthesis pathway and determines fruit skin colour in sweet cherry. Interestingly, we identified three functional alleles of the gene causally leading to the different colours at mature stage. Meanwhile, our experimental results of yeast two‐hybrid assays and chromatin immunoprecipitation assays revealed that Pav MYB10.1 might interact with proteins Pavb HLH and Pav WD40, and bind to the promoter regions of the anthocyanin biosynthesis genes Pav ANS and Pav UFGT ; these findings provided to a certain extent mechanistic insight into the gene's functions. Additionally, genetic and molecular evidences confirmed that Pav MYB10.1 is a reliable DNA molecular marker to select fruit skin colour in sweet cherry.