Analyzing barley gene expression profiles in response to abiotic stress is critical to understanding how barley manages stress, and provides vital information to improve environmental stress tolerance for stable crop production. We developed an Agilent 60-mer oligo DNA microarray with 42,491 probe sets based on the sequences of 36,632 barley (Hordeum vulgare L.) full-length cDNA clones and conducted global expression profiling on barley seedlings subjected to desiccation, salt, cold and abscisic acid (ABA). We identified 281 genes that were differentially expressed in response to desiccation, salt, and cold stresses and ABA treatment. Among them, a class C heat shock transcription factor (HvHsfC1) and a homeodomain leucine zipper (HD-Zip) family I transcription factor (HvHox22) showed more than tenfold and fourfold higher expression, respectively, in response to the stimuli. Heat shock and HD-Zip transcription factors function as important regulators in stress responses in rice (Oryza sativa) and Arabidopsis thaliana; our results suggest these two transcription factors also play important roles in abiotic stress responses in barley. We mapped HvHox22 to the long arm of chromosome 2H and HvHsfC1 to the long arm of 4H, where drought resistance quantitative trait loci were previously detected. Our microarray data and identification of these stress response genes provide key information for dissection of the mechanism of abiotic stress tolerance in barley.