Sorbitol is synthesized in Rosaceae species, especially in source organs, in a pathway involving aldose 6-phosphate reductase (A6PR, EC 1.1.1.200). As compatible solutes, sorbitol and other sugar alcohols assist in the ability of the plant to withstand abiotic stress conditions. Here, we identify two tandemly-duplicated genes in a non-Rosaceae species (Arabidopsis thaliana L.), and show that the proteins encoded by At2g21250 (AtA6PR1) and At2g21260 (AtA6PR2) possess the molecular characteristics of A6PRs. Consistent with bioinformatic predictions, we determined that green fluorescent protein-tagged versions of AtA6PR1 and AtA6PR2 are cytosolically localized, a finding supported by immunoblotting using a specific anti-AtA6PR1 antisera after subcellular fractionation of Arabidopsis leaves. We also show that under standard growth conditions, both genes are widely-expressed, whilst AtA6PR1 protein accumulates in both source and sink organs. Under short term (24 h) cold (4 °C) or saline (150 mM NaCl) stress, both genes respond differentially and reciprocally in leaf and root tissues, suggesting a sub-functionalization of their roles. The identification of homozygous T-DNA insertional ata6pr1-mutants will facilitate the functional characterization of this gene, and help to determine its role under abiotic stress conditions.