Accurate patient setup and target localization are essential to advanced radiation therapy treatment. Significant improvement has been made recently with the development of image-guided radiation therapy, in which image guidance facilitates short treatment course and high dose per fraction radiotherapy, aiming at improving tumor control and quality of life. Many imaging modalities are being investigated, including x-ray computed tomography (CT), ultrasound imaging, positron emission tomography, magnetic resonant imaging, magnetic resonant spectroscopic imaging, and kV/MV imaging with flat panel detectors. These developments provide unique imaging techniques and methods for patient setup and target localization. Some of them are different; some are complementary. This paper reviews the currently available kV x-ray CT systems used in the radiation treatment room, with a focus on the CT-on-rails systems, which are diagnostic CT scanners moving on rails installed in the treatment room. We will describe the system hardware including configurations, specifications, operation principles, and functionality. We will review software development for image fusion, structure recognition, deformation correction, target localization, and alignment. Issues related to the clinical implementation of in-room CT techniques in routine procedures are discussed, including acceptance testing and quality assurance. Clinical applications of the in-room CT systems for patient setup, target localization, and adaptive therapy are also reviewed for advanced radiotherapy treatments.