Introduction Data on omega-3 polyunsaturated fatty acids in relation to cardiovascular disease are limited in women. The aim of this study was to examine longitudinal relations of tuna and dark fish, α-linolenic acid, and marine omega-3 fatty acid intake with incident major cardiovascular disease in women. Methods This was a prospective cohort study of U.S. women participating in the Women’s Health Study from 1993 to 2014, during which the data were collected and analyzed. A total of 39,876 women who were aged ≥45 years and free of cardiovascular disease at baseline provided dietary data on food frequency questionnaires. Analyses used Cox proportional hazards models to evaluate the association between fish and energy-adjusted omega-3 polyunsaturated fatty acid intake and the risk of major cardiovascular disease, defined as a composite outcome of myocardial infarction, stroke, and cardiovascular death, in 38,392 women in the final analytic sample (96%). Results During 713,559 person years of follow-up, 1,941 cases of incident major cardiovascular disease were confirmed. Tuna and dark fish intake was not associated with the risk of incident major cardiovascular disease ( p -trend >0.05). Neither α-linolenic acid nor marine omega-3 fatty acid intake was associated with major cardiovascular disease or with individual cardiovascular outcomes (all p -trend >0.05). There was no effect modification by age, BMI, or baseline history of hypertension. Conclusions In this cohort of women without history of cardiovascular disease, intakes of tuna and dark fish, α-linolenic acid, and marine omega-3 fatty acids were not associated with risk of major cardiovascular disease.