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Heterosis was defined as the advantage of hybrid performance over its parents in terms of growth and productivity. Previous studies showed that differential gene expression between hybrids and their parents is responsible for the heterosis; however, information on systematic identification and characterization of the differentially expressed genes are limited. In this study, an interspecific hybrid between common wheat (Triticum aestivum. L., 2n = 6x = 42, AABBDD) line 3338 and spelt (Triticum spelta L. 2n = 6x = 42, AABBDD) line 2463 was found to be highly heterotic in both aerial growth and root related traits, and was then used for expression assay. A modified suppression subtractive hybridization (SSH) was used to generate four subtracted cDNA libraries, and 748 nonreduandant cDNAs were obtained, among which 465 had high sequence similarity to the GenBank entries and represent diverse of functional categories, such as metabolism, cell growth and maintenance, signal transduction, photosynthesis, response to stress, transcription regulation and others. The expression patterns of 68.2% SSH-derived cDNAs were confirmed by reverse Northern blot, and semi-quantitative RT-PCR exhibited the similar results (72.2%). And it was concluded that the genes differentially expressed between hybrids and their parents involved in diverse physiological process pathway, which might be responsible for the observed heterosis.