Evidence regarding the role of dairy fat intake in cardiovascular disease (CVD) has been mixed and inconclusive. Most earlier studies have used self-reported measures of dietary intake and focused on relatively racially homogeneous populations. Circulating biomarkers of dairy fat in a multiethnic cohort provide objective measures of dairy fat intake and facilitate conclusions relevant to populations with different diets and susceptibility to CVD. In a multiethnic cohort of 2837 US adults aged 45 to 84 years at baseline (2000-2002), phospholipid fatty acids including 15:0, 14:0, and trans-16:1n7 were measured using standardized methods, and the incidence of CVD prospectively adjudicated. Self-reported whole-fat dairy and butter intakes had strongest associations with 15:0, rather than 14:0 or trans-16:1n7. In multivariate models including demographics and lifestyle and dietary habits, each SD-unit of 15:0 was associated with 19% lower CVD risk (hazard ratio [95% CI] 0.81 [0.68 to 0.98]) and 26% lower coronary heart disease (CHD) risk (0.74 [0.60 to 0.92]). Associations were strengthened after mutual adjustment for 14:0 and trans-16:1n-7 and were similar after adjustment for potential mediators. Plasma phospholipid 14:0 and trans-16:1n-7 were not significantly associated with incident CVD or CHD. All findings were similar in white, black, Hispanic, and Chinese American participants. Plasma phospholipid 15:0, a biomarker of dairy fat, was inversely associated with incident CVD and CHD, while no association was found with phospholipid 14:0 and trans-16:1n-7. These findings support the need for further investigation of CVD effects of dairy fat, dairy-specific fatty acids, and dairy products in general.