The genus Prunus includes several species producing edible drupes of significant economic importance widely grown around the world, such as peach, nectarine, plum, prune, apricot, almond and cherry. The recent sequencing of the complete genome of peach, considered the reference Prunus genome, together with the availability of new high-throughput sequencing technologies offers new possibilities for Prunus genome and transcriptome analysis. RNA-Seq involves direct sequencing of cDNAs (from RNA) using high-throughput sequencing technologies, allowing   the analysis of the level of transcription from a particular genomic region or a whole genome. Thus far, RNA-Seq experiments in plants and Prunus have been oriented primarily toward the high-throughput sequencing of cDNA from messenger RNA (mRNA-Seq). High-throughput micro-RNA sequencing (miRNA-Seq) is another component of the global application of RNA-Seq, but has fewer applications in plants and it is  being applied to Prunus. mRNA-Seq represents the latest and most powerful tool for characterizing transcriptomes. The application of these mRNA-Seq technologies offers new opportunities in the development of efficient marker-assisted selection strategies in Prunus breeding through the study of RNA regulation at the transcriptional level. In this work, the design of mRNA-Seq experiments in Prunus species is described and discussed. This description includes details of experimental design, sequencing technology, type of analysis and existent bioinformatics tools. In the case of mRNA, the following two major analysis approaches are described: mapping reads to the reference genome and de novo transcriptome assembly. These analyses will lead to other more complex analyses, including expressed sequence tag (ESTs) generation, single nucleotide polymorphism (SNPs) identification, alternative splicing analysis and differential expression analysis.