Objective: Ankylosing spondylitis (AS) is associated with an increased risk of cardiovascular disease (CVD). Microvasculature changes can precede overt CVD, but have been studied poorly in AS. The retinal vasculature is easily accessible and changes are associated with CVD (e.g. arteriolar narrowing, venular widening, loss of tortuosity). This proof of concept study compared the retinal microvasculature of AS patients with healthy controls, and the influence of gender. Methods: Cross-sectional case-control study comparing AS patients with healthy controls. Main inclusion criteria were: age 50–75 years, no diabetes mellitus and, for AS, fulfillment of the modified New York criteria. All subjects underwent fundus photography, analyzed with Singapore I Vessel Assessment software, and Optical Coherence Tomography Angiography (OCTA). Subjects were compared with generalized estimating equations (GEE). Multivariable analyses were adjusted for demographics and cardiovascular risk, and stratified for gender. Results: Fifty-nine AS patients and 105 controls were included (50% women). Controls were significantly older than patients (68 versus 60, p<0.01), but did not differ in cardiovascular profile. Patients had a lower retinal arteriolar tortuosity (β ̶-0.1, 95%CI [-0.2; -0.01], p = 0.02), and higher vessel density (β 0.5, 95% CI [0.1; 0.9], p = 0.02). In addition, male AS patients showed a lower arteriovenular ratio compared to male controls (β -0.03, p = 0.04, 95%CI [-0.05; -0.001]). There were no differences found between women with and without AS. Conclusion: This study detected several retinal microvascular changes, in AS patients compared to controls, which have been associated with CVD. Retinal imaging might be an interesting tool for future CVD screening.