The universal stress proteins (USPs) play an important role in enhancing survival rate during prolonged exposure to heat shock, nutrient starvation, or stressors from agents that arrest cell growth or damage DNA structures. Searching the HarvEST database of barley resulted in 25 putative USP cDNA sequences. Of these, 16 could translate into intact proteins (putative USPs). The alignments of multiple amino acid sequences between the putative barley USPs with those of Arabidopsis and Methanococcus jannaschii resulted in a set of common residues involved in ATP-binding. The 16 putative USPs in barley and the 21 in Arabidopsis were clustered into seven groups, which were distinct from those of E. coli. The genes in these different groups have different intron/exon structures. Nine putative USP genes of barley were cloned successfully based on their sequence characteristics, and they contain two or three introns each. Two of these introns were present in all the genes, one located between beta2 and alpha2, and the other between beta 4 and alpha 4. Five sets of primers were successfully developed for these putative USP genes. Two of them were mapped on chromosome 1H and the other three were located on three different chromosomes, 2H, 3H and 6H, respectively. Expression analyses were carried out for nine of these putative USP genes. The expression for two of them was undetectable within 27 h following exposure to salt stress. Six of the other seven were expressed in both root and leaf, and the remaining one was expressed in root only. The majority of these genes was expressed more in the salt-sensitive variety, Morex, than in the more tolerant variety, Steptoe.