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Summary: Background: Lacunar stroke accounts for a quarter of cases of acute ischaemic stroke; however, its underlying pathophysiology remains unclear. Our aim was to establish whether there is an association between changes in the retinal microvasculature and lacunar stroke that might provide clues to the pathology of cerebral small vessel disease. Methods: In this cross-sectional study, we recruited patients who presented with acute stroke at three centres in two countries (Sydney and Melbourne, Australia, and Singapore). Each patient had standardised clinical assessments, retinal photography, and CT or MRI of the brain. Changes in the retinal microvasculature were assessed from retinal photographs by graders who were masked to the patients'' clinical details. Lacunar stroke was diagnosed according to a modified version of the TOAST criteria (Treatment of Acute Stroke Trial) or the OCSP criteria (Oxfordshire Community Stroke Project) and by MRI findings. Findings: We recruited 1321 patients aged 19 to 94 years with acute ischaemic stroke; 410 (31%) had lacunar stroke. Patients with acute lacunar stroke were no more likely to have hypertension (p=0·12), diabetes (p=0·51), or hypercholesterolaemia (p=0·91) than were patients with other types of ischaemic stroke. However, patients with lacunar stroke were more likely to have retinal microvessel signs, particularly when stroke subtype was confirmed using diffusion-weighted MRI, than were patients with other stroke subtypes. After adjustment for age, sex, study site, smoking history, hypertension, and diabetes, the patients with lacunar stroke were more likely than those with other stroke subtypes to have microvessel signs, and when stroke subtype was confirmed by diffusion-weighted MRI the odds ratios were: 3·55 (95% CI 1·77–7·12) for focal arteriolar narrowing; 1·96 (1·19–3·24) for arteriovenous nipping; 2·32 (1·42–3·79) for enhanced light reflex of the arteriolar wall; 1·33 (0·74–2·41) for generalised retinal arteriolar narrowing; 1·45 (0·84–2·51) for small retinal arteriole:venule ratio; and 1·35 (0·80–2·26) for retinal venular widening. Interpretation: Our findings suggest that acute lacunar stroke is a manifestation of non-atherothrombotic occlusive small vessel disease, which might have implications for the prevention and treatment of this stroke subtype. Funding: National Health and Medical Research Council of Australia; National Medical Research Council of Singapore; Scottish Funding Council; New South Wales Health. [Copyright &y& Elsevier]