We aimed to investigate whether fish and long chain omega-3 polyunsaturated fatty acid (LCn-3 PUFA) consumption changed appreciably during adolescence. We also assessed whether these dietary variables are associated with retinal microvascular signs (possible markers of future cardiovascular disease risk). 633 children had dietary data at ages 12 and 17. Fish and LCn-3 PUFA [eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA)] intake was assessed by a food frequency questionnaire. Retinal vessel caliber was quantified from digital photographs using computer software. Mean energy-Adjusted intakes (mg/day) of total LCn-3 PUFA, EPA, and DHA at age 12 were 297.1 ±261.1; 102.5±106.9; and 129.7±137.7, respectively; and this increased significantly at age 17 to: 347.0±324.0 (p<0.0001); 122.5±132.7 (p = 0.0001); and 160.3±171.4 (p <0.0001), respectively. Increasing quartiles of LCn-3PUFA intake were associated with widening of mean retinal arteriolar caliber among 17-year old girls ∼3.9 μm (multivariable-Adjusted Ptrend = 0.001). Girls who consumed ≥2 serves of fish/week versus those who did not had ∼2.1 μm wider retinal arterioles (p = 0.03). No associations were observed among boys or with retinal venules. Mean dietary intakes of LCn-3 PUFA increased during adolescence, but are still below recommended levels of consumption. These results suggest that LCn-3 PUFA and fish intake might have a beneficial influence.