The purpose of this study was to determine interfractional variation of the centroid position and volume of internal target volume (ITV) during stereotactic body radiation therapy (SBRT) of lung cancer. From January 2014 to August 2014, a total of 32 patients with 37 primary or metastatic lung tumors were enrolled in our study. All patients received SBRT treatment in 4–5 fractions to a median dose of 48 Gy. Both 3D CT and 4D CT scans were used for radiotherapy treatment planning. 3D CBCT was acquired prior to treatment delivery to verify patient positioning. A total of 163 3D CBCT images were available for evaluation. 3D CBCT scans acquired for verification were registered with simulation CT scans. The ITVs were contoured on all verification 3D CBCT scans and compared to the initial gross target volume (GTV) or ITV in treatment planning system. GTV was based on 3D CT while ITV was based on both 3D CT and 4D CT. To assess the interfractional variation of ITV centroid position, we used vertebrae body adjacent to the tumor as reference point when performing the registration procedure. To eliminate the effect of time on tumor volume between simulation CT scan and the first fraction, the interfractional variation of ITV was evaluated from the first fraction to the last fraction. The overall 3D vector shift was 4.4±2.5 mm (range: 0.4−13.8 mm). The interfractional variation of ITV centroid position in superior‐inferior, anterior‐posterior, and left‐right directions were −0.7±2.7 mm, −1.4±3.4 mm, and −0.5±2.2 mm, respectively. No significant difference was observed between three directions (p=0.147). Large interfractional variations (≥5 mm) were observed in 12 fractions (9.3%) in superior‐inferior direction, 24 fractions (18.6%) in anterior‐posterior direction, and 5 fractions (3.9%) in left‐right direction. No time trend of tumor volume change measured in 3D CBCT was detected during four fractions (p=0.074). A significant (p=0.010) time trend was detected when evaluating the time trend of ITV change during 5 fractions and diameter was found to be significantly correlated with the ITV change (p=0.000). ITV did not show significant regression during SBRT treatment, but interfractional variation in the ITV centroid position was observed, especially in anterior‐posterior direction. An isotropic margin of 7 mm around ITV might be necessary for adequate coverage of interfractional variation of ITV centroid position, but only in case no soft tissue‐based setup is performed during SBRT treatment.PACS number(s): 87.55.dk