AbstractInter-fractional tumor variance would lead to insufficient dosage or overdose in tumor region during lung cancer radiotherapy. However, previous works have not considered influence of inter-fractional tumor amplitude variance at treatment position due to lack of effective evaluation method during radiotherapy, especially for lung tumor within the lower lobe. Our objective was to investigate inter-fractional tumor baseline shift and amplitude variance due to respiratory motion with 4DCBCT simulation and guidance during stereotactic ablative body radiotherapy (SABR) for lung tumor. Subject included 19 patients with lung tumor within the lower lobe. 4DCBCT-simulated images at treatment position were acquired sequentially to determine internal tumor volume (ITV) and reference tumor motion at simulation process. Compared with reference tumor motion, 95 4DCBCT-guided images were acquired during each treatment to evaluate inter-fractional tumor baseline shift and amplitude variance, which were − 0.0 ± 1.3 mm and − 0.2 ± 1.4 mm in left–right(LR) direction, 0.9 ± 2.3 mm and 0.4 ± 2.9 mm in superior-inferior (SI) direction, 0.1 ± 1.5 mm and − 0.4 ± 2.0 mm in anterior–posterior (AP) direction. ITV margin were 3.5 mm, 7.5 mm and 5.3 mm in LR, SI and AP directions with van Herk’s (Int J Radiat Oncol Biol Phys 52(5):1407–1422, 2002) formula. 4DCBCT simulation and guidance is a reliable method to evaluate inter-fractional tumor variance during SABR for lung tumor within the lower lobe. ITV margin of 3.5 mm, 7.5 mm and 5.3 mm in LR, SI and AP directions would ensure greater tumor coverage during SABR for lung tumor within the lower lobe.