AIMS/HYPOTHESIS: Changes in retinal microvasculature may reflect insulin resistance. We examined the association of changes in retinal microvasculature during pregnancy and risk of subsequent abnormal glucose metabolism in a cohort of mothers at baseline and 5 years postpartum. METHODS: Of the participants from the Singapore birth cohort (Growing Up in Singapore Towards Healthy Outcomes [GUSTO]), 276 mothers attended both baseline (at 26-28 weeks of gestation) and follow-up (5 year postpartum) visits. At baseline we performed retinal photography and assessed retinal microvascular variables using a validated grading system. At follow-up, we assessed glucose tolerance using a 75 g OGTT. We defined abnormal glucose metabolism if participants: (1) had onset of gestational diabetes mellitus (GDM) in subsequent pregnancies within a 5 year follow-up period (n = 103) or (2) had prediabetes (impaired fasting glucose, impaired glucose tolerance or HbA1c 5.7-6.4% [39-46 mmol/mol]) and diabetes diagnosed at the 5 year follow-up visit (n = 84), according to WHO guidelines. RESULTS: The incidence of GDM in subsequent pregnancy and abnormal glucose metabolism 5 years postpartum was 25.2% and 30.4%, respectively. Each 10 μm widening in retinal venular calibre was associated with a significant risk of postpartum abnormal glucose metabolism (RR 1.2 [95% CI 1.0, 1.5]), independent of maternal age, college education, ethnicity, pre-pregnancy BMI and GDM at baseline. Narrower retinal arteriolar calibre and venular branching angle at baseline was associated with a higher insulin resistance index (1.4 [95% CI 1.1, 1.7] and 1.3 [95% CI 1.1, 1.6], respectively) at follow-up. CONCLUSIONS/INTERPRETATION: Retinal microvasculature in pregnant women was associated with abnormal glucose metabolism 5 years postpartum. Alteration of microvascular structure during pregnancy may signal subclinical changes that underlie the development of prediabetes and diabetes.