Purpose There are few data on the effect of serum lipids on microvascular disease. This study assessed the relationships between serum lipid levels and microvascular disease, as seen in the retina, among participants who attended a population-based study in Australia (n ¼ 3654, aged 49 þ years). Methods Diameters of retinal arterioles and venules were measured from digitised photographs of each participant to obtain an estimate of generalised arteriolar narrowing. Focal arteriolar narrowing, arteriovenous nicking, and retinopathy lesions (microaneurysms, haemorrhages, hard/soft exudates) were graded using a standard protocol. Fasting blood tests were performed in 89% of subjects. Adjusted means were calculated using general linear models. Logistic regression models were used to determine the odds ratios for retinal microvascular signs. Results After controlling for age, sex, body mass index, smoking, and mean arterial blood pressure, elevated high-density lipoprotein cholesterol was associated with narrower retinal arterioles (P trend ¼ 0.002) and venules (P trend ¼ 0.03) and with increased odds of generalised arteriolar narrowing (odds ratio 1.6, 95% confidence interval 1.1-2.2 for the highest vs the lowest quintile of high-density lipoprotein cholesterol). Serum triglyceride had a U-shaped relationship with venular diameter (P trend ¼ 0.003). We found no consistent pattern of association between serum total cholesterol or low-density lipoprotein cholesterol and any retinal microvascular signs. Conclusions These findings suggest that microvascular disease in the retina may result from processes distinct from dyslipidaemia.