This paper presents the design evolution, fabrication, and testing of a novel patient and organ-specific, three-dimensional (3D)-printed phantom for external beam radiation therapy (EBRT) of prostate cancer. In contrast to those found in current practice, this phantom can be used to plan and validate treatment tailored to an individual patient. It contains a model of the prostate gland with a dominant intraprostatic lesion (DIL), seminal vesicles, urethra, ejaculatory duct, neurovascular bundles, rectal wall, and penile bulb generated from a series of combined T2-weighted/dynamic contrast-enhanced magnetic resonance (MR) images. The iterative process for designing the phantom based on user interaction and evaluation is described. Using the CyberKnife System at Boston Medical Center, a treatment plan was successfully created and delivered. Dosage delivery results were validated through gamma index calculations based on radiochromic film measurements which yielded a 99.8% passing rate. This phantom is a demonstration of a methodology for incorporating high-contrast MR imaging into computed-tomography-based radiotherapy treatment planning; moreover, it can be used to perform quality assurance (QA).