Background: Polyploid breeding is a powerful approach for Populus genetic improvement because polyploid trees have valuable characteristics, including better timber quality and a higher degree of stress resistance compared with their full-sib diploids. However, the genetic mechanism underlying this phenomenon remains unknown.Objective: To better understand the proteomic changes between Populus allotriploids and diploids, we examined the proteomic profiles of allotriploid and diploid Populus by iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDI-TOF/TOF mass spectrometry (MS). Method: iTRAQ labeling coupled with two-dimensional liquid chromatography and MALDITOF/ TOF mass spectrometry (MS). Results: Between the Populus allotriploid and the full-sib diploid, 932 differentially expressed proteins (DEPs) were identified. These DEPs were primarily involved in stress, defense, transportation, transcriptional and/or translational modification, and energy production. The pathway analysis indicated that most of the DEPs were implicated in carbohydrate transport and metabolism, nitrogen metabolism and glycolysis, and the ribosome assembly pathway. These data suggest high protein divergence between Populus allotriploids and diploids, and rapid changes during hybridization. Conclusion: The results provide new data for further understanding of the mechanisms of polyploid trees that generally display increased height growth compared with their full-sib diploids.