Single center retrospective cohort study. We performed a retrospective study evaluating the incidence and degree of L4-5 anterior spondylolisthesis in patients with standard supine MRI, standing flexion-extension radiographs, and prone CT. We hypothesize that prone CT imaging will provide greater sensitivity for instability compared with conventional flexion extension or supine positions. Dynamic lumbar instability evaluated by flexion-extension radiographs may underestimate the degree of lumbar spondylolisthesis. Despite efforts to characterize dynamic instability, significant variability remains in current guidelines regarding the most appropriate imaging modalities to adequately evaluate instability. We assessed single-level (L4-5) anterolisthesis between 2014 and 2022 with standing lateral conventional radiographs (CR), flexion-extension images, prone CT images (CT), or supine MRI images (MRI). We identified 102 patients with L4-5 anterolisthesis. The average translation (±SD) measured were 4.9±2.2mm (CR), 2.5±2.6mm (CT), and 3.7±2.6mm (MRI) (P<0.001). The mean difference in anterolisthesis among imaging modalities was 2.7±1.8mm between CR and CT (P<0.001), 1.8±1.4mm between CR and MRI (P<0.001), and 1.6±1.4mm between CT and MRI (P=0.252). Ninety-two of 102 patients (90.2%) showed greater anterolisthesis on CR compared with CT, 72 of 102 (70.6%) comparing CR to MRI, and 27 of 102 (26.5%) comparing CT to MRI. We found that 17.6% of patients exhibited ≥3mm anterior translation comparing CR with MRI, whereas 38.2% of patients were identified comparing CR with CT imaging (χ2 test P=0.0009, post hoc Fisher exact test P=0.0006 between CR and CT). Only 5.9% of patients had comparable degrees of instability between flexion-standing. Prone CT imaging revealed the greatest degree of single L4-5 segmental instability compared with flexion-extension radiographs.