Background To investigate the mechanisms of low back pain (LBP) triggered by the five-repetition sit-to-stand test (5R-STS test) in degenerative lumbar spondylolisthesis (DLS) from radiographic perspective, as well as to determine the most useful diagnostic modalities in the evaluation of segmental instability. Methods We retrospectively performed a study of 78 patients (23 men and 55 women) with symptomatic DLS at L4/5 in our institution between April 2020 and December 2021. Each patient was assessed by using the 5R-STS test and received a series of radiographs including the upright standing, natural sitting, standing flexion-extension radiographs, and supine sagittal MRI. Enrolled patients were divided into two groups based on the 5R-STS test score: severe group and mild group. Translational and angular motion was determined by comparing nature sitting radiograph (S) with upright standing radiograph (N) (Combined, SN), flexion/extension radiographs (FE) as well as nature sitting radiograph (S) with a supine sagittal MRI image (U) (Combined, SU). Results Overall, 78 patients were enrolled, and there were 31(39.7%) patients in group S and 47(60.3%) patients in group M, with an average age of 60.7 ± 8.4 years. The natural sitting radiograph demonstrated the maximum slip percentage (SP) and the highest kyphotic angle both in group S and group M. Compared with group M, group S revealed significantly higher SP in the natural sitting position (24.1 vs 19.6; p = 0.002). The lumbar slip angular in group S with a sitting position was significantly higher than that in group M (-5.2 vs -1.3; p < 0.001). All patients in group S had OFI and 28 patients of them were diagnosed with lumbar instability by using the combination of nature sitting radiograph (S) and supine sagittal MRI image (U) (Combined, SU). Conclusion DLS patients with positive sign of the 5R-STS test is a distinct subgroup associated with lumbar instability. The modality of the combination of nature sitting radiograph (S) and supine MRI (U) had a significant advantage in terms of the ability to identify segmental instability.