Objective To investigate the correlation between mutiple-voxel magnetic resonance spectroscopy (1H-MRS) parameter choline/creatine (Cho/Cr) and distribution of glioma stem cells (GSCs). Methods Sixteen patients with high-grade glioma approved by pathology, admitted to our hospital form August 2012 and March 2015, were enrolled in our study. They were performed 1H-MRS before surgery, and apparently different regions of Cho/Cr were identified. With the help of intraoperative neuronavigation, different Cho/Cr tissue samples were gained accurately (Cho/Cr hypermetabolism group and Cho/Cr hypometabolism group). The different distribution of glioma stem cells in glioma tissues of the two groups was detected via neurosphere culture; immunohistochemistry and Western blotting were employed to detect the CD133 and nestin expressions. Results Neurospheres were successfully cultured from different glioma tissues, and the sphere formation rate from Cho/Cr hypermetabolism group was significantly higher as compared with that from Cho/Cr hypometabolism group (13.94±3.55 vs. 8.04± 1.47, P<0.05). The immunohistochemistry results indicated that the expressions of CD133 and nestin in the Cho/Cr hypermetabolism group were significantly higher as compared with those in the Cho/Cr hypometabolism group ([22.96±2.28]% vs. [18.04±1.36]%, [25.47±2.43]% vs. [19.74±1.66]%, P<0.05). Western blotting showed that the relative protein expressions of CD133 and nestin in the Cho/Cr hypermetabolism group were significantly higher as compared with those in the Cho/Cr hypometabolism group (0.50±0.17 vs. 0.30±0.08, 0.45±0.13 vs. 0.27±0.07, P<0.05); and the protein expressions of CD133 and nestin were positively correlated with Cho/Cr (r=0.972, P=0.000; r=0.762, P=0.000). Conclusion 1H-MRS parameter Cho/Cr reveals the distribution differences of cancer stem cells in high-grade gliomas, which can assist in finding and resecting the glioma stem cells-rich region.