With the advent of fast imaging hardware and specialized software, additional non-invasive magnetic resonance characterization of tumors has become available through proton magnetic resonance spectroscopy (MRS), hemodynamic imaging and diffusion-weighted imaging (DWI). Thus, patterns could be discerned to discriminate different types of tumors and even to infer their possible evolution in time. The purpose of this study was to investigate the correlation between MRS, DWI, histopathology and Ki-67 labeling index in a large number of brain tumors. Localized proton spectra were obtained in 47 patients with brain tumors who subsequently underwent surgery (biopsy or tumor removal). We performed MRS with short echo-time (30 ms) and metabolic values in spectra were measured using an external software with 25 peaks. In all patients who had DWI, we measured apparent diffusion coefficients (ADC) in the same region of interest (ROI) where the voxel in MRS was located. In most tumors the histological diagnosis and Ki-67 labeling index had been determined on our original surgical specimen. Cho/Cr, (Lip+Mm)/Cr, NAA/(Cho+Cr) and Glx/Cr indexes in MRS allowed discriminating between low- and high-grade gliomas and metastases (MTs). Likewise, absolute ADC values differentiated low- from high-grade gliomas expressed by Ki-67 labeling index. A novel finding was that high Glx/Cr in vivo MRS index (similar to other known indexes) was a good predictor of tumor grading.