We aimed to precisely estimate intra-tumoral heterogeneity using spatially regularized spectral clustering (SRSC) on multiparametric MRI data and compare the efficacy of SRSC with the previously reported segmentation techniques in MRI studies. Six NMRI nu/nu mice bearing subcutaneous human glioblastoma U87 MG tumors were scanned using a dedicated small animal 7T magnetic resonance imaging (MRI) scanner. The data consisted of T2 weighted images, apparent diffusion coefficient maps, and pre- and post-contrast T2 and T2* maps. Following each scan, the tumors were excised into 2-3-mm thin slices parallel to the axial field of view and processed for histological staining. The MRI data were segmented using SRSC, K-means, fuzzy C-means, and Gaussian mixture modeling to estimate the fractional population of necrotic, peri-necrotic, and viable regions and validated with the fractional population obtained from histology. While the aforementioned methods overestimated peri-necrotic and underestimated viable fractions, SRSC accurately predicted the fractional population of all three tumor tissue types and exhibited strong correlations (r(necrotic) = 0.92, r(peri-necrotic) = 0.82 and r(viable) = 0.98) with the histology. The precise identification of necrotic, peri-necrotic and viable areas using SRSC may greatly assist in cancer treatment planning and add a new dimension to MRI-guided tumor biopsy procedures.