Abstract Purpose: The purpose of this paper is to quantify if using an average intensity projection (AIP) scan or a 30% phase (mid-ventilation surrogate, MidV) scan as the reference image for patient position verification affects reproducibility of lung cancer patient alignment under free-breathing cone beam computed tomography (CBCT) image guidance and to analyze the effects of common clinical issues on registration variability. Methods: AIPs were retrospectively created for 16 lung patients (14 SBRT, 2 conventional treatments) originally planned/treated using the 30% phase MidV surrogate scan as reference. The study included 3-5 CBCTs from each patient. Registrations were performed between the AIP-CBCT and between the MidV-CBCT by 5 individuals (student, medical physics resident, medical resident, medical physicist, and attending physician) using MIM 6.2 image registration platform (Beachwood, OH). The images were rigidly registered, internal tumor volume (ITV) contours were displayed, and no rotational adjustments were allowed to reflect real treatment conditions. Additionally, the registrations for AIP-CBCT and MidV-CBCT were repeated 3 times by one individual for intra-observer variability assessment. Patient setup rotations, tumor volume, tumor motion, and breathing variability were estimated for correlation with registration variability. Results: The magnitude of the average intra-observer standard deviations from the lateral (LAT), anterior-posterior (AP), and superior-inferior (SI) directions for the AIP/CBCT and MidV/CBCT registrations were 0.9 mm and 1.2 mm, respectively. The magnitude of the average inter-observer standard deviations for the AIP/CBCT and MidV/CBCT were 1.7 mm and 1.8 mm, respectively. Average discrepancies over the whole population were found to be small; however, some individual patients presented high variability. Patient-specific cases with high variability were analyzed and observations on its potential causes are discussed. Conclusion: The differences in alignment using AIP versus MidV as the reference images are, when averaged over the population studied, very small and clinically irrelevant for PTV margins > 5mm; however, individual patients may be impacted in a clinically relevant manner if smaller margins, 3 mm and below, are used instead.