Hyperhydricity is a syndrome that causes morpho-physiological malformations in tissue culture plantlets. Micro-RNAs (miRNA) are small non-coding RNAs that play important regulatory roles in plant development, stress response, and adaptation to environmental conditions. In this study, differential expression analysis indicated that miRNAs play an underlying role in the responses to the hyperhydricity syndrome in peach Prunus persica (L.) leaves. 24 known and three novel potential miRNAs were characterized in hyperhydric and non-hyperhydric transcriptome libraries. The miRNA-target transcript analyses indicated that transport, plant cuticle development, intracellular part, and stress response are regulated by miRNAs in hyperhydric leaves. It is also suggested that miR5021 and miRnovel2 might play critical regulatory roles in hyperhydricity regarding miRNA-based response to stress. This study went one step further to advance understanding of molecular miRNA-based regulatory mechanisms regarding responses to hyperhydricity in peach.