Prostatic neoplasms are not uniformly distributed within the prostate volume. With recent developments in three-dimensional intensity-modulated and image-guided radiation therapy, it is possible to treat different volumes within the prostate to different thresholds of doses. This approach has the potential to adapt the dose to the biologic aggressiveness of various clusters of tumor cells within the gland. The definition of tumor burden volume in prostate cancer can be facilitated by the use of magnetic resonance spectroscopy (MRS). The increasing sensitivity and specificity of MRS to the prostate is causing new interest in its potential role in the definition of target subvolumes at higher risk of failure following radical radiotherapy. Prostate MRS might also play a role as a noninvasive predictive factor for tumor response and treatment outcome. We review the use of MRS in radiation therapy for prostate cancer by evaluating its accuracy in the classification of aggressive cancer regions and target definition; its current role in the radiotherapy planning process, with special interest in technical issues behind the successful inclusion of MRS in clinical use; and available early experiences as a prognostic tool.