Using microarray analysis, we identified regulatory and signaling-related genes with differential expression in three genotypes with varying degrees of salt tolerance, Triticum aestivum , the amphiploid, and the wheat substitution line DS3E(3A). Lophopyrum elongatum is among one of the most salt-tolerant members of the Triticeae; important genetic stocks developed from crosses between wheat and L. elongatum provide a unique opportunity to compare gene expression in response to salt stress between these highly related species. The octaploid amphiploid contains the entire genome of T. aestivum and L. elongatum, and the disomic substitution line DS3E(3A) has chromosome 3A of wheat replaced by chromosome 3E of L. elongatum. In this study, microarray analysis was used to characterize gene expression profiles in the roots of three genotypes, Triticum aestivum, the octaploid amphiploid, and the wheat DS3E(3A) substitution line, in response to salt stress. We first examined changes in gene expression in wheat over a time course of 3 days of salt stress, and then compared changes in gene expression in wheat, the T. aestivum × L. elongatum amphiploid, and in the DS3E(3A) substitution line after 3 days of salt stress. In the time course experiment, 237 genes had 1.5 fold or greater change at least one out of three time points assayed in the experiment. The comparison between the three genotypes revealed 304 genes with significant differences in changes of expression between the genotypes. Forty-two of these genes had at least a twofold change in expression in response to salt treatment; 18 of these genes have signaling or regulatory function. Genes with significant differences in induction or repression between genotypes included transcription factors, protein kinases, ubiquitin ligases and genes related to phospholipid signaling.