The diagnosis and therapy of childhood brain tumors, most of which are low grade, can be complicated because of their frequent adjacent location to crucial structures, which limits diagnostic biopsy. Also, although new prognostic biomarkers identified by molecular analysis or DNA microarray gene profiling are promising, they too depend on invasive biopsy. Here, we test the hypothesis that combining information from biologically important intracellular molecules (biomarkers), noninvasively obtained by proton magnetic resonance spectroscopic imaging, will increase the diagnostic accuracy in determining the clinical grade of pediatric brain tumors. We evaluate the proton magnetic resonance spectroscopic imaging exams for 66 children with brain tumors. The intracellular biomarkers for choline-containing compounds (Cho), N-acetylaspartate, total creatine, and lipids and/or lactate were measured at the highest Cho region and normalized to the surrounding healthy tissue total creatine. Neuropathological grading was done with WHO criteria. Normalized Cho and lipids and/or lactate were elevated in high-grade (n = 23) versus low-grade (n = 43) tumors, which multiple logistic regression confirmed are independent predictors of tumor grade (for Cho, odds ratio 24.8, P < 0.001; and for lipids and/or lactate, odds ratio 4.4, P < 0.001). A linear combination of normalized Cho and lipids and/or lactate that maximizes diagnostic accuracy was calculated by maximizing the area under the receiver operating characteristic curve. Proton magnetic resonance spectroscopic imaging, although not a proxy for histology, provides noninvasive, in vivo biomarkers for predicting clinical grades of pediatric brain tumors.