To approach heterogeneity of tumor tissues, we examined proteomes of normal and tumor tissues from colorectal cancer patients by laser microdissection and 2D-DIGE with saturation dye and alkaline range IPG gel. By distinguishing the tumor cells based on tissue localization, we found that a range of protein spots showed different intensity between normal and diseased tissues. Subsequent mass spectrometric protein identification identified the proteins for protein spots. We found that the protein spots from identical proteins showed the different tissue localization in colorectal cancer. These results highlight the potential utilities of laser microdissection, 2D-DIGE, and immobilized pH gradient gel with alkaline range.