High-throughput DNA and RNA sequencing technologies have resulted in the successful identification of Single nucleotide polymorphisms (SNPs). In order to develop a large SNP set for wide application in apricot (Prunus armeniaca L.), we carried out RNA high-throughput sequencing (RNA-Seq) in two apricot genotypes, “Rojo Pasión” and “Z506-7.” After trimming and cleaning, 70 % of RNA-Seq reads were aligned to the reference peach genome. Sequences uniquely mapped on the peach genome allowed for the discovery of 300 k SNP/INDEL variations, with a density of one SNP per 850 bp. Some 95 SNPs of the 99 tested were analyzed in a set of 37 apricot accessions using SNPlex™ genotyping technology. The results provide accurate values for nucleotide diversity in coding sequences in apricot. The combination of a highly efficient RNA-Seq approach and SNPlex™ high-throughput genotyping technology thus provide a powerful tool for apricot genetic analysis. SNP markers produced a total of 267 allelic combinations in the 37 apricot accessions assayed with a mean of 2.8 combinations per locus, an observed heterozygosity per marker ranging from 0.06 to 0.65, and a power of discrimination ranging from 0.12 to 0.66. In addition, SNP markers confirmed parentage and also determined relationships between the accessions in a manner consistent with their pedigree relationships.