Carotenoids are naturally occurring tetraterpenoids that play a key role in fruit coloration, and yellow peaches are one of the best sources of carotenoid intake. MYB transcription factors are one of the largest families in plants and play an important role in the regulation of plant secondary metabolite biosynthesis. However, peach MYB family genes have not been fully analyzed, and in particular, MYBs that regulate carotenoid biosynthesis have not been fully characterized. In this study, 190 peach MYB genes, containing 68 1R-MYBs, 118 2R-MYBs, 3 3R-MYBs, and 1 4R-MYB, were identified at the genome level using bioinformatics methods. These 190 MYBs were classified into 27 subfamilies based on their phylogenetic relationships with Arabidopsis thaliana MYB family members, and they were unevenly distributed across eight chromosomes. MYB genes of the same subfamily exhibit similar but not identical gene structures and conserved motifs. The promoter regions contain cis-acting elements associated with stress response, hormone response, and plant growth and development. There were 54 collinear pairs of MYB genes in the peach genome, compared with 233 and 221 collinear pairs with Rosa chinensis and Arabidopsis, respectively. Thirteen differentially expressed genes in the carotenoid biosynthesis pathway in yellow peach were identified by transcriptome sequencing and contained MYB binding sites on their promoters. Based on a phylogenetic analysis, we identified 13 PpMYBs that may be involved in carotenoid biosynthesis, and a correlation analysis revealed that they regulate carotenoid accumulation by positively or negatively regulating the expression of carotenoid biosynthetic genes. Further degradome sequencing screened that mdm-miR858 was able to target PpMYB17 and PpMYB126 involved in the regulation of carotenoid biosynthesis. Our findings provide new insights into the potential role of MYB transcription factors in carotenoid biosynthesis and provide a theoretical basis for their molecular mechanisms.