Changes in cold hardiness were confirmed with relative electrolyte leakage (REL) method in the shoots of two peach cultivars (Prunus persica Janghowon Hwangdo and Odoroki) during cold acclimation and deacclimation. Changes in proline (Pro) content and related gene expressions were also analyzed. Particularly, transcript accumulations of P5CS and P5CR were examined using quantitative real-time RT-PCR. REL in the shoots of two peach cultivars was significantly different during the entire experimental period. Cold hardiness of two cultivars increased gradually to December 2012, and then decreased to April 2013, whereas Pro contents of ‘Janghowon Hwangdo’ and ‘Odoroki’ were reduced from the beginning of the experiment to February 2012 and then increased in the spring. Interestingly, P5CS gene encoding an enzyme, which catalyzes conversion from glutamic acid (Glu) into glutamic-γ-semialdehyde (GSA) in the first step of Pro pathway, showed the contrasting patterns with Pro contents of two cultivars. On the other hand, P5CR gene encoding an enzyme, which catalyzes conversion from Δ1-pyrroline-5-carboxylate(P5C)intoPro in the final step of Pro pathway, showed the similar patterns to Pro contents in two cultivars. Our results demonstrate that Pro responds negatively to low temperatures in the shoot of different peach cultivars, including the supplemental 10 peach cultivars, and expression of both P5CS and P5CR genes could show contrasting patterns from each other. Our results suggest that identification of both P5CS and P5CR genes are required necessarily for accurate analysis of Pro biosynthesis because Pro accumulation is affected more by expression of P5CR gene.