Abstract: Objective To investigate the set-up errors in lung cancer patients by kilo-voltage cone beam CT (kvCBCT) technology, and to provide the reference data for clinical target volume (CTV) to planning target volume (PTV) in radiotherapy. Methods Forty-one patients with lung cancer were divided randomly into two groups: Group A (20 cases) were treated with Synergy linear accelerator, and Group B (21 cases) were treated with Trilogy TX linear accelerator. All patients underwent kvCBCT before radiotherapy and every week during the treatment as needed. Set-up errors of kvCBCT were calculated according to its matched and planned CT images in left-right(X), superior-inferior(Y) and anterior-posterior(Z) axes. Difference of set-up errors was compared between two accelerators and margin of planning target volume (MPTV) was calculated. Results The set-up errors of Group A and B in X, Y and Z axes were (1.79±0.15) mm and (2.16±0.17) mm, (3.71±0.28) mm and (4.29±0.32) mm, (1.66±0.15) mm and (1.96±0.18) mm, respectively. Set-up errors in three axes in Group A were less than those in Group B(P>0.05). MPTV of Group A and B in X, Y and Z axes were 4.36 mm and 4.94 mm, 8.22 mm and 9.20 mm, 4.38 mm and 5.02 mm, respectively. Conclusion Application of kvCBCT could significantly reduce the set-up errors of radiotherapy in patients with lung cancer.