Purpose To study the inter- and intrafraction tumor reproducibility with active breath control (ABC) utilizing cone-beam computed tomography (CBCT), and compare validity of registration with two different regions of interest (ROI). Methods and materials Thirty-one lung tumors in 19 patients received conventional or stereotactic body radiotherapy with ABC. During each treatment, patients had three CBCT scanned before and after online position correction and after treatment. These CBCT images were aligned to the planning CT using the gray scale registration of tumor and bony registration of the thorax, and tumor position uncertainties were then determined. Results The interfraction systematic and random translation errors in the left–right (LR), superior–inferior (SI) and anterior–posterior (AP) directions were 3.6, 4.8, and 2.9 mm; 2.5, 4.5, and 3.5 mm, respectively, with gray scale alignment; 1.9, 4.3, 2.0 mm and 2.5, 4.4, 2.9 mm, respectively, with bony alignment. The interfraction systematic and random rotation errors with gray scale and bony alignment groups ranged from 1.4° to 3.0° and 0.8° to 2.3°, respectively. The intrafraction systematic and random errors with gray scale registration in LR, SI, AP directions were 0.9, 2.0, 1.8 mm and 1.5, 1.7, 2.9 mm, respectively, for translation; 1.5°, 0.9°, 1.0° and 1.2°, 2.2°, 1.8°, respectively, for rotation. The translational errors in SI direction with bony alignment were significantly larger than that of gray scale ( p < 0.05). Conclusions With CBCT guided online correction the interfraction positioning errors can be markedly reduced. The intrafraction errors were not diminished by the use of ABC. Rotation errors were not very remarkable both inter- and intrafraction. Gray scale alignment of tumor may provide a better registration in SI direction.