To investigate the underlying intratumoral diversity of molecular profiles in glioblastomas, a proteomic approach was introduced to compare samples from regions of different histological grade. Using two-dimensional gel electrophoresis (2DE) with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we compared prospectively collected tissue samples of different histological grade areas of three glioblastoma patients. Consistent results showing relatively high expression of ubiquitin carboxyl-terminal esterase L1 in low-histological-grade areas (Grade 2 > Grades 3 and 4) and high expression of transthyretin in high-histological-grade areas (Grade 2 < Grades 3 and 4) were demonstrated. These results were confirmed by western blot (WB) analysis and immunohistochemical staining. This study provided the evidence of multifarious proteomic signatures according to regional and histological heterogeneity in glioblastomas.