MicroRNAs are a class of small, endogenous, non-coding RNA molecules that negatively regulate gene expression at the transcriptional or the post-transcriptional level. Although a large number of miRNAs have been identified in many plant species, especially from model plants and crops, they remain largely unknown in peach. In this study, 110 potential miRNAs belonging to 37 families were identified using computational methods. A total of 43 potential targets were found for 21 families based on near-perfect or perfect complementarity between the plant miRNA and the target sequences. A majority of the targets were transcription factors which play important roles in peach development. qRT-PCR analysis of RNA samples prepared from different peach tissues for 25 miRNA families revealed that miRNAs were differentially expressed in different tissues. Furthermore, two target genes were experimentally verified by detection of the miRNA-mediated mRNA cleavage sites in peach using RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE). Finally, we studied the expression pattern of the two target genes in three different tissues of peach to further understand the mechanism of the interaction between miRNAs and their target genes.