PURPOSE: In brain gliomas, non-invasive biomarkers reflecting tumor cellularity would be useful to guide supramarginal resections and to plan stereotactic biopsies. We aim to validate a previously-trained machine learning algorithm that generates cellularity prediction maps (CPM) from multiparametric MRI data to an independent, retrospective external cohort of gliomas undergoing image-guided biopsies, and to compare the performance of CPM and diffusion MRI apparent diffusion coefficient (ADC) in predicting cellularity. METHODS: A cohort of patients with treatment-naïve or recurrent gliomas were prospectively studied. All patients underwent pre-surgical MRI according to the standardized brain tumor imaging protocol. The surgical sampling site was planned based on image-guided biopsy targets and tissue was stained with hematoxylin-eosin for cell density count. The correlation between MRI-derived CPM values and histological cellularity, and between ADC and histological cellularity, was evaluated both assuming independent observations and accounting for non-independent observations. RESULTS: Sixty-six samples from twenty-seven patients were collected. Thirteen patients had treatment-naïve tumors and fourteen had recurrent lesions. CPM value accurately predicted histological cellularity in treatment-naïve patients (b = 1.4, R = 0.2, p = 0.009, rho = 0.41, p = 0.016, RMSE = 1503 cell/mm), but not in the recurrent sub-cohort. Similarly, ADC values showed a significant association with histological cellularity only in treatment-naive patients (b = 1.3, R = 0.22, p = 0.007; rho = -0.37, p = 0.03), not statistically different from the CPM correlation. These findings were confirmed with statistical tests accounting for non-independent observations. CONCLUSION: MRI-derived machine learning generated cellularity prediction maps (CPM) enabled a non-invasive evaluation of tumor cellularity in treatment-naïve glioma patients, although CPM did not clearly outperform ADC alone in this cohort.