Hyperhydricity is a morphophysiological disorder of plants in tissue culture characterized morphologically by the presence of translucent, thick, curled, and fragile leaves as a result of excessive water intake. Since clonal propagation is a major in vitro technique for multiplying plants vegetatively, the emergence of hyperhydricity-related symptoms causes significant economic losses to agriculture and horticulture. Although numerous efforts have been hitherto devoted to the morphological and anatomical responses of plants to hyperhydricity, the underlying molecular mechanism remains largely unknown. Here, a genome-wide transcriptome analysis was performed to identify differentially expressed genes in hyperhydric and nonhyperhydric leaves of peach [Prunus persica (L.) Batsch]. The RNA sequencing (RNA-Seq) analysis showed that the expression of >300 transcripts was altered between control and hyperhydric leaf cells. The top 30 differentially expressed transcripts (DETs) were related to the posttranscriptional regulators of organelle gene expression and photosynthesis, cellular elimination, plant cuticle development, and abiotic stress response processes. The expression of 10 DETs was also conformed by quantitative real-time polymerase chain reaction (RT-qPCR) in hyperhydric and nonhyperhydric leaves. As a complex biological process, hyperhydricity alters the expression of various transcripts including transcription factor (Myb2), RNA binding protein (pentatricopeptide, PPR), transporter protein (ABC), and Laccase3. Thus, this genome-wide transcriptome profiling study may help elucidate the molecular mechanism of hyperhydricity.