Background: This study was conducted to compare differences in imaging features and clinical symptoms between patients with single-level isthmic spondylolisthesis (IS) at L4 and at L5 and to investigate the correlation between imaging and clinical parameters. Methods: This cross-sectional study evaluated patients with single-level IS who were enrolled between June 2011 and June 2018. A total of 139 patients, 44 in the L4 IS group and 95 in the L5 IS group, met the study criteria and were included. Imaging and clinical parameters obtained from the two groups were compared and analyzed. Results: Patients in the L4 IS group had smaller lower lumbar lordosis (LLL) (27.1 ± 8.2 vs. 30.9 ± 9.3, P = 0.021) and were of older age (58.5 ± 8.7 vs. 52.8 ± 10.1, P < 0.01) than those in the L5 IS group. As per the Roussouly classification system, most patients with L4 IS were classified as Type 2 (43.2%), whereas most patients with L5 IS fell under Type 3 (44.2%). In the L5 IS group, pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), lumbar lordosis (LL), and L5 incidence (L5I) were positively associated with slippage rate (SR), but the lumbosacral angle (LSA) was negatively associated with SR (P < 0.01). In the L4 IS group, only L5I showed a positive association with SR (P < 0.01). More significant associations were found among sagittal lumbo-pelvic parameters in the L5 IS group, but none were found between SR and Oswestry Disability Index (ODI) in either group. Conclusions: When compared with patients with L5 IS, patients with L4 IS were of older age and had straighter low lumbar curvature when they were obviously symptomatic. PI was an important parameter for patients with L5 IS while for those with L4 IS, L5I deserved more attention for its significantly positive correlation with the degree of slippage.