DWI showed great potential for estimation of histopathological and molecular profile of human glioma. 97 patients with suspected glioma underwent pre-operative MRI-scans, including high b-value DWI. ADC-maps from pairs of two b-values were calculated. Post-interventional histopathological tumor grading was realized on a molecular basis using the molecular markers IDH-mutation, 1p/19q- and ATRX-loss. Significant differences (p < 0.001) were found between oligodendroglioma, astrozytoma and GBM. Best discrimination was achieved when calculating the ADC-maps from b-values of 500 and 2500 s/mm2. Therefore, ADC-map based evaluation of glioma in DWI provides great potential in accurate pre-interventional diagnosing of glioma subtypes.