Prunus salicina is one of the most economically important stone fruits. However, there is scarce genetic information available, which makes it difficult to implement marker-assisted selection (MAS) in genetic improvement programs. Recently, next-generation sequencing has greatly enhanced breeding program strategies, generating information associated with the identification of expressed sequence tag–simple sequence repeats (EST–SSRs) and single-nucleotide polymorphisms (SNPs), two of the most used molecular markers in MAS. Few studies have focused on developing EST–SSR markers considering both gene expression levels of contrasting phenotypes and specific transcription factors of metabolic pathways. This study investigated the transcriptome profile of P. salicina in fruits with contrasting skin colors, obtaining 54,224 unique contigs. From this dataset, 44 EST–SSRs have been generated, considering gene expression levels of contrasting phenotypes and specific transcription factor from three metabolic pathways: citric acid, carbohydrate metabolism and flavonoid pathways. Three EST–SSR markers developed from the putative flavonoid pathway transcription factors PsMYB10, PsMYB1 and PsbHLH35 were selected to determine genetic structure in 29 cultivars. This structure was contrasted with the genetic structure generated using genomic SNPs obtained by genotyping-by-sequencing (GBS). The analysis using SNPs identified two groups, while the use of selected EST–SSRs identified three. In contrast to the structure given by the SNPs, the EST–SSRs grouped all the yellow cultivars in one cluster, which was composed mainly of cultivars of this color. The EST–SSRs developed in this study may be considered as candidate markers to be evaluated in MAS strategies in genetic improvement programs.