AIM: To analyze differences in ultra-widefield fluorescein angiography (UWFA) findings between dynamic and static images of eyes with diabetic retinopathy (DR). METHODS: This cross-sectional study included 28 eyes of 28 patients with DR undergoing UWFA. A series of UWFA images acquired from each patient were converted into a time-lapse video and used as a dynamic image. A single, clear, arteriovenous phase image was chosen as a static image. Non-perfusion index (NPI) and its correlation with vascular abnormalities in different zones were compared between dynamic and static UWFA imaging. RESULTS: NPI appeared to increase from the center to the far-periphery in both groups. Dynamic NPI was lower in the total retinal area (0.26 vs 0.29, P=0.009) and far-periphery (0.33 vs 0.36, adjusted P=0.042), which was contrary to the static NPI. Far-peripheral NPI was associated with intraretinal microvascular abnormality in the posterior area in both groups. CONCLUSION: Time-lapse dynamic UWFA imaging is a useful modality to differentially diagnose hypofluorescence in the most peripheral region. This modality could provide a reliable method for NPI measurement.