In Peach (Prunus persica) several physiological changes, such as woolliness, triggered by chilling injury are involved in major production losses due to cold storage of the fruits during shipping. Additionally, the low level of polymorphisms among peach varieties is an important limitation in the search for new molecular markers that could be associated with economically important traits. Therefore, a functional approach was employed to associate candidate genes with an informative marker in peach. The data was obtained from the results of an in silico analysis of four different cold peach treatments. Thirty two candidate genes were selected that were aligned against Arabidopsis thaliana genomic sequences to design intron-flanking EST-PCR markers. These markers were used to position the candidate genes on the Prunus genetic reference map. In the physiological response to chilling injury, cell wall integrity, carbohydrate metabolism and stress response pathways could be involved, therefore candidate genes associated by Gene Ontology annotation to these pathways were included in the analysis. The designed markers were positioned to the Texas X Earlygold (TxE) genetic reference map through selective mapping methodology (Bin mapping). 72% of these new markers showed polymorphism in the TxE Binset population and 31% of them were successfully mapped to a genetic position on the Prunus reference map. The bioinformatic methodology used in this work includes a first approach in search for functional molecular markers associated to differentially expressed genes under certain physiological condition which in addition to the Bin mapping approach allows addressing a genetically anchored position to these new markers.