AIM: To evaluate a high-resolution functional imaging device that yields quantitative data regarding macular blood flow and capillary network features in eyes with diabetic retinopathy (DR). METHODS: Prospective, cross-sectional comparative case-series in which blood flow velocities (BFVs) and non-invasive capillary perfusion maps (nCPMs) in macular vessels were measured in patients with DR and in healthy controls using the Retinal Functional Imager (RFI) device. RESULTS: A total of 27 eyes of 21 subjects were studied [9 eyes nonproliferative diabetic retinopathy (NPDR), 9 eyes proliferative diabetic retinopathy (PDR) and 9 controls]. All diabetic patients were type 2. All patients with NPDR and 5 eyes with PDR also had diabetic macular edema (DME). The NPDR group included eyes with severe (n=3) and moderate NPDR (n=6), and were symptomatic. A significant decrease in venular BFVs was observed in the macular region of PDR eyes when compared to controls (2.61±0.6 mm/s and 2.92±0.72 mm/s in PDR and controls, respectively, P=0.019) as well as PDR eyes with DME compared to NPDR eyes (2.36±0.51 mm/s and 2.94±1.09 mm/s in PDR with DME and NPDR, respectively, P=0.01). CONCLUSION: The RFI, a non-invasive imaging tool, provides high-resolution functional imaging of the retinal microvasculature and quantitative measurement of BFVs in visually impaired DR patients. The isolated diminish venular BFVs in PDR eyes compared to healthy eyes and PDR eyes with DME in comparison to NPDR eyes may indicate the possibility of more retinal vein compromise than suspected in advanced DR.