In response to environmental cues plants undergo changes in gene expression that result in the up- or down-regulation of specific genes. To identify genes in peach [Prunus persica (L.) Batsch.] trees whose transcript levels are specifically affected by low temperature (LT) or short day photoperiod (SD), we have created suppression subtractive hybridization (SSH) libraries from bark tissues sampled from trees kept at 5 °C and 25 °C under short day (SD) photoperiod or exposed to a night break (NB) interruption during the dark period of the SD cycle to simulate a long day (LD) photoperiod. Sequences expressed in forward and reverse subtractions using various subtracted combinations of temperature and photoperiod treatments were cloned, sequenced, and identified by BLAST and ClustalW analysis. Low temperature treatment resulted in the up-regulation of a number of cold-responsive and stress-related genes and suppression of genes involved in “housekeeping” functions (e.g., cell division and photosynthesis). Some stress-related genes not observed to be up-regulated under LT were increased in response to SD photoperiod treatments. Comparison of the patterns of expression as a consequence of different temperature and photoperiod treatments allowed us to determine the qualitative contribution of each treatment to the regulation of specific genes.