This is the first study of proteomics of wheat bunt fungi Tilletia controversa (TCK), T. caries (TCT), and T. foetida (TFL) using the iTRAQ technique. Based on the relative quantities of specific proteins between each two pathogens, we found 50 up-regulated and 80 down-regulated protein genes in TCK compared to TFL, 62 up-regulated and 82 down-regulated protein genes in TCT compared to TFL, 47 up-regulated and 30 down-regulated protein genes in TCK compared to TCT, and there were 1 protein of up-regulated and 4 proteins of down-regulated in the three pairs. These protein data could be of great value for exploring the key proteins which play an important role in the interactions of these pathogens with their host. Some of them could be valuable for differentiating the three pathogens with monoclonal antibodies produced by the specific proteins and may enable in-site detection of the pathogens and performing routine monitoring as a diagnostic assay in wheat shipments.