Stereotactic body radiotherapy for prostate cancer is rapidly growing in popularity. Stereotactic body radiotherapy plans mimic those of high-dose rate brachytherapy, with tight margins and inhomogeneous dose distributions. The impact of interfraction anatomical changes on the dose received by organs at risk under these conditions has not been well documented. To estimate anatomical variation during stereotactic body radiotherapy, 10 patients were identified who received a prostate boost using robotic stereotactic body radiotherapy after completing 25 fractions of pelvic radiotherapy with daily megavoltage computed tomography. Rectal and bladder volumes were delineated on each megavoltage computed tomography, and the stereotactic body radiotherapy boost plan was registered to each megavoltage computed tomography image using a point-based rigid registration with 3 fiducial markers placed in the prostate. The volume of rectum and bladder receiving 75% of the prescription dose (V75%) was measured for each megavoltage computed tomography. The rectal V75% from the daily megavoltage computed tomographies was significantly greater than the planned V75% (median increase of 0.93 cm 3 , P < .001), whereas the bladder V75% on megavoltage computed tomography was not significantly changed (median decrease of À0.12 cm 3 , P ¼ .57). Although daily prostate rotation was significantly correlated with bladder V75% (Spearman r ¼ .21, P ¼ .023), there was no association between rotation and rectal V75% or between prostate deformation and either rectal or bladder V75%. Planning organ-at-risk volume-based replanning techniques using either a 6-mm isotropic expansion of the plan rectal contour or a 1-cm expansion from the planning target volume in the superior and posterior directions demonstrated significantly improved rectal V75% on daily megavoltage computed tomographies compared to the original stereotactic body radiotherapy plan, without compromising plan quality. Thus, despite tight margins and full translational and rotational corrections provided by robotic stereotactic body radiotherapy, we find that interfraction anatomical variations can lead to a substantial increase in delivered rectal doses during prostate stereotactic body radiotherapy. A planning organ-at-risk volumebased approach to treatment planning may help mitigate the impact of daily organ motion and reduce the risk of rectal toxicity.