Purpose To investigate the feasibility of a three‐dimensional amide‐proton‐transfer (APT) imaging sequence with gradient‐ and spin‐echo readouts at 3 Tesla in patients with high‐ or low‐grade gliomas. Materials and Methods Fourteen patients with newly diagnosed gliomas were recruited. After B0 inhomogeneity correction on a voxel‐by‐voxel basis, APT‐weighted images were reconstructed using a magnetization‐transfer‐ratio asymmetry at offsets of ±3.5 ppm with respect to the water resonance. Analysis of variance post hoc tests were used for statistical evaluations, and results were validated with pathology. Results In six patients with gadolinium‐enhancing high‐grade gliomas, enhancing tumors on the postcontrast T1‐weighted images were consistently hyperintense on the APT‐weighted images. Increased APT‐weighted signal intensity was also clearly visible in two pathologically proven, high‐grade gliomas without gadolinium enhancement. The average APT‐weighted signal was significantly higher in the lesions than in the contralateral normal‐appearing brain tissue (P < 0.001). In six low‐grade gliomas, including two with gadolinium enhancement, APT‐weighted imaging showed iso‐intensity or mild punctate hyperintensity within all the lesions, which was significantly lower than that seen in the high‐grade gliomas (P < 0.001). Conclusion The proposed three‐dimensional APT imaging sequence can be incorporated into standard brain MRI protocols for patients with malignant gliomas. J. Magn. Reson. Imaging 2013;38:1119–1128. © 2013 Wiley Periodicals, Inc.