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BACKGROUND In Cone-Beam Computed Tomography (CBCT) guided radiotherapy, off-by-one vertebral body misalignments are rare but serious errors which lead to wrong-site treatments. PURPOSE An automatic error detection algorithm was developed that uses a three-branch convolutional neural network error detection model (EDM) to detect off-by-one vertebral body misalignments using planning computed tomography (CT) images and setup CBCT images. METHODS Algorithm training and test data consisted of planning CTs and CBCTs from 480 patients undergoing radiotherapy treatment in the thoracic and abdominal regions at two radiotherapy clinics. The clinically applied registration was used to derive true-negative (no error) data. The setup and planning images were then misaligned by one vertebral body in both the superior and inferior directions, simulating the most likely misalignment scenarios. For each of the aligned and misaligned 3D image pairs, 2D slice pairs were automatically extracted in each anatomical plane about a point within the vertebral column. The three slice pairs obtained were then inputted to the EDM which returned a probability of vertebral misalignment. One model (EDM1 ) was trained solely on data from institution #1. EDM1 was further trained using a lower learning rate on a dataset from institution #2 to produce a fine-tuned model, EDM2 . Another model, EDM3 , was trained from scratch using a training dataset composed of data from both institutions. These three models were validated on a randomly selected and unseen dataset composed of images from both institutions, for a total of 303 image pairs. The model performances were quantified using a receiver operating characteristic analysis. Due to the rarity of vertebral body misalignments in the clinic, a minimum threshold value yielding a specificity of at least 99% was selected. Using this threshold, the sensitivity was calculated for each model, on each institution's test set separately. RESULTS When applied to the combined test set, EDM1 , EDM2 , and EDM3 resulted in an area under curve of 99.5%, 99.4% and 99.5%, respectively. EDM1 achieved a sensitivity of 96% and 88% on Institution #1 and Institution #2 test set, respectively. EDM2 obtained a sensitivity of 95% on each institution's test set. EDM3 achieved a sensitivity of 95% and 88% on Institution #1 and Institution #2 test set, respectively. CONCLUSION The proposed algorithm demonstrated accuracy in identifying off-by-one vertebral body misalignments in CBCT-guided radiotherapy that was sufficiently high to allow for practical implementation. It was found that fine-tuning the model on a multi-facility dataset can further enhance the generalizability of the algorithm. This article is protected by copyright. All rights reserved.