A 27-bp variable number tandem repeat (VNTR) in intron 4 of endothelial nitric oxide synthase (eNOS) gene has been associated with the risk for developing diabetic retinopathy (DR) in various ethnic populations. Hundred and eighty seven patients with retinopathy (cases; DR+) and 188 patients without retinopathy (controls: DR-) from southern India who had type 2 diabetes mellitus (T2DM) for more than 10 years, were included in the study. We could neither find significant allelic association with clinical severity of DR nor with macular edema. Our results suggest lack of association of intron 4 VNTR of eNOS gene with DR in southern India.