Abstract Aims/hypothesis The aim of the study was to examine the association of retinal vessel morphometry with BP, body composition and biochemistry, and to determine whether these associations differ by diabetes status. Methods The UK Biobank ocular assessment included 68,550 participants aged 40-70 years who underwent non-mydriatic retinal photography, BP and body composition measurements, and haematological analysis. A fully automated image analysis program provided measurements of retinal vessel diameter and tortuosity. The associations between retinal vessel morphology and cardiometabolic risk factors by diabetes status were examined using multilevel linear regression, to provide absolute differences in vessel diameter and percentage differences in tortuosity (allowing for within-person clustering). Results A total of 50,233 participants (a reduction from 68,550) were included in these analyses. Overall, those with diabetes had significantly more tortuous venules and wider arteriolar diameters compared with those without. Associations between venular tortuosity and cardiometabolic risk factors differed according to diabetes status (p interaction <0.01) for total fat mass index, HbA_1c, C-reactive protein, white cell count and granulocyte count. For example, a unit rise in white cell count was associated with a 0.18% increase (95% CI 0.05, 0.32%) in venular tortuosity for those without diabetes and a 1.48% increase (95% CI 0.90, 2.07%) among those with diabetes. For arteriolar diameter, significant interactions were evident for systolic BP, diastolic BP, mean arterial pressure (MAP) and LDL-cholesterol. For example, a 10 mmHg rise in systolic BP was associated with a −0.92 μm difference (95% CI −0.96 to −0.88 μm) in arteriolar diameter for those without diabetes, and a −0.58 μm difference (95% CI −0.76 to −0.41 μm) among those with diabetes. No interactions were observed for arteriolar tortuosity or venular diameters. Conclusions/interpretation We provide clear evidence of the modifying effect of diabetes on cardiometabolic risk factor associations with retinal microvascular architecture. These observations suggest the occurrence of preclinical disease processes, and may be a sign of impaired autoregulation due to hyperglycaemia, which has been suggested to play a pivotal role in the development of diabetes-related microvascular complications. Data Availability The data supporting the results reported here are available through the UK Biobank (https://www.ukbiobank.ac.uk/enable-your-research/apply-for-access). Graphical abstract