Accumulation of anthocyanin pigments is a primary determinant of fruit quality in peach ( Prunus persica L. Batsch). The recessive blood-flesh trait (bf), identified in French cultivars has been first mapped on chromosome 4. However, the underlying gene has yet been identified. In this study, the genetic, molecular and transcriptional bases of the bf trait were investigated. Blood-flesh cultivars showed high levels of cyanidin-3-glucoside and cyanidin-3-rutinoside in the fruit mesocarp about 45–60 days after anthesis. A fine mapping coupled to a positional cloning strategy mapped the bf locus to a 80-kb interval on peach linkage group (LG) 4. The sequence polymorphism analysis of genes present in this interval allowed the identification of only one candidate gene for bf as being a deletion of 21-bp in exon 3 of a PSAK-like gene. A specific marker of the deletion was developed and validated in various genetic backgrounds. An expression study of PpPSAK as well as structural, regulatory and photosynthesis related genes was performed on a kinetic from 60 days after blooming up to fruit maturity. Overexpression of PpPSAK in flesh and midrib of bf fruit was correlated with expression of anthocyanin biosynthesis structural genes. Interactions between proteins were observed and open discussion on functional pathways.