The Omega-3 Index, a measure of long-chain omega-3 fats in red blood cell membranes, predicts heart disease mortality in adults, but its association with cardiovascular risk factors in younger populations is unknown. We determined the Omega-3 Index in adolescents participating in the Western Australian Pregnancy (Raine) Cohort, assessed associations with diet, lifestyle and socioeconomic factors, and investigated independent associations with cardiovascular and metabolic risk factors. Red blood cell fatty acid analysis was determined for 1,301 adolescents aged 13–15 years. Risk factors examined were blood pressure, fasting blood insulin and glucose concentrations, and fasting blood lipids including ratios. The mean Omega-3 Index was 4.90 ± 1.04% (range 1.41–8.42%). When compared with categories identified in adults, 15.6% of adolescents were in the high risk category (Index < 4%). Age (P < 0.01), maternal education (P < 0.01) and BMI (P = 0.05) were positively associated with the Omega-3 Index. The Index was positively associated with dietary intakes of eicosapentaenoic and docosahexaenoic acid (P < 0.01), protein (P < 0.01), omega-3 fats (P < 0.04), and food groups of fish and wholegrains (both P < 0.01), and negatively associated with intakes of soft drinks and crisps (both P < 0.01). In boys, the Omega-3 Index was independently associated with total (β = 0.06, P = 0.01) and HDL-cholesterol (β = 0.03, P = 0.01), and diastolic blood pressure (β = −0.68, P = 0.04). The predictability of the Index for the risk of cardiovascular disease later in life warrants further investigation in the adolescent population.