To better understand the development of wheat roots, a reference map of the major soluble proteins of wheat roots was established using a combination of 2-DE and MALDI TOF MS and MS/MS, and a total of 450 protein spots were detected with silver staining in a pH ranges of 4-7, of which 282 spots corresponding to 240 proteins were identified. These identified proteins were grouped into diverse functional categories. In comparison with a wheat leave proteome, in root, proteins involved in metabolism and transport were over-represented, whereas proteins involved in energy, disease and defense, transcription, and signal transduction were under-represented. To further get an insight into the molecular basis of wheat heterosis, differential proteome analysis between hybrid and parents were performed. A total of 45 differentially expressed protein spots were detected, and both quantitative and qualitative differences could be observed. Moreover, 25 of the 45 differentially expressed protein spots were identified, which were involved in metabolism, signal transduction, energy, cell growth and division, disease and defense, secondary metabolism. These results indicated that hybridization between two parental lines can cause expression differences between wheat hybrid and its parents not only at mRNA levels but also at protein abundances.