The purpose of this study is to investigate the feasibility of using a continuous-time random-walk (CTRW) diffusion model, together with a quartile histogram analysis, for assessing glioma malignancy by probing tissue heterogeneity as well as cellularity. In this prospective study, 91 patients (40 females, 51 males) with histopathologically proven gliomas underwent MRI at 3 T. The cohort included 42 grade II (GrII), 19 grade III (GrIII) and 29 grade IV (GrIV) gliomas. Echo-planar diffusion-weighted imaging was conducted using 17 b-values (0-4000 s/mm(2)). Three CTRW model parameters, including an anomalous diffusion coefficient D-m, and two parameters related to temporal and spatial diffusion heterogeneity alpha and beta, respectively, were obtained. The mean parameter values within the tumor regions of interest (ROIs) were computed by utilizing the first quartile of the histograms as well as the full ROI for comparison. A Bonferroni-Holm-corrected Mann-Whitney U-test was used for the group comparisons. Individual and combinations of the CTRW parameters were evaluated for the characterization of gliomas with a receiver operating characteristic analysis. All first-quartile mean CTRW parameters yielded significant differences (p-values < 0.05) between pair-wise comparisons of GrII (D-m: 1.14 +/- 0.37 mu m(2)/ms; alpha: 0.904 +/- 0.03, beta: 0.913 +/- 0.06), GrIII (D-m: 0.88 +/- 0.21 mu m(2)/ms; alpha: 0.888 +/- 0.01, beta: 0.857 +/- 0.06) and GrIV gliomas (D-m: 0.73 +/- 0.22 mu m(2)/ms; alpha: 0.878 +/- 0.01; beta: 0.791 +/- 0.07). The highest sensitivity, specificity, accuracy and area-under-the-curve of using the combinations of the first-quartile parameters were 84.2%, 78.5%, 75.4% and 0.76 for GrII and GrIII classification; 86.2%, 89.4%, 75% and 0.76 for GrIII and GrIV classification; and 86.2%, 85.7%, 84.5% and 0.90 for GrII and GrIV classification, respectively. Quartile-based analysis produced higher accuracy and area-under-the-curve than the full ROI-based analysis in all classifications. The CTRW diffusion model, together with a quartile-based histogram analysis, offers a new way for probing tumor structural heterogeneity at a subvoxel level, and has potential for in vivo assessment of glioma malignancy to complement histopathology.