Pectins are synthesized and secreted to the cell wall as highly methyl-esterified polymers and demethyl-esterified by pectin methylesterases (PMEs), which are regulated by pectin methylesterase inhibitors (PMEIs). PMEs and PMEIs are involved in pectin degradation during fruit softening; however, the roles of the PME and PMEI gene families during fruit softening remain unclear. Here, 71 PME and 30 PMEI genes were identified in the peach (Prunus persica) genome and shown to be unevenly distributed on all eight chromosomes. The 71 PME genes comprised 36 Type-1 PMEs and 35 Type-2 PMEs. Transcriptome analysis showed that 11 PME and 15 PMEI genes were expressed during fruit ripening in melting flesh (MF) and stony-hard (SH) peaches. Three PME and five PMEI genes were expressed at higher levels in MF than in SH fruit and exhibited softening-associated expression patterns. Upstream regulatory cis elements of these genes related to hormone response, especially naphthaleneacetic acid and ethylene, were investigated. One PME (Prupe.7G192800) and two PMEIs (Prupe.1G114500 and Prupe.2G279800), and their promoters were identified as potential targets for future studies on the biochemical metabolism and regulation of fruit ripening. The comprehensive data generated in this study will improve our understanding of the PME and PMEI gene families in peach. However, further detailed investigation is necessary to elucidate the biochemical function and regulation mechanism of the PME and PMEI genes during peach fruit ripening.