BACKGROUND: Dyslipidemia is an independent risk factor for coronary heart disease (CHD). Standard lipid panel cannot capture the complexity of the blood lipidome (ie, all molecular lipids in the blood). To date, very few large-scale epidemiological studies have assessed the full spectrum of the blood lipidome on risk of CHD, especially in a longitudinal setting. METHODS AND RESULTS: Using an untargeted liquid chromatography-mass spectrometry, we repeatedly measured 1542 lipid species from 1835 unique American Indian participants who attended 2 clinical visits (≈5.5 years apart) and followed up to 17.8 years in the Strong Heart Family Study (SHFS). We first identified baseline lipid species associated with risk of CHD, followed by replication in a European population. The model adjusted for age, sex, body mass index, smoking, hypertension, diabetes, low-density lipoprotein cholesterol, estimated glomerular filtration rate, education, and physical activity at baseline. We then examined the longitudinal association between changes in lipid species and changes in cardiovascular risk factors during follow-up. Multiple testing was controlled by the false discovery rate. We found that baseline levels of multiple lipid species (eg, phosphatidylcholines, phosphatidylethanolamines, and ceramides) were associated with the risk of CHD and improved the prediction accuracy over conventional risk factors in American Indian people. Some identified lipids in American Indian people were replicated in European people. Longitudinal changes in multiple lipid species (eg, acylcarnitines, phosphatidylcholines, and triacylglycerols) were associated with changes in cardiovascular risk factors. CONCLUSIONS: Baseline plasma lipids and their longitudinal changes over time are associated with risk of CHD. These findings provide novel insights into the role of dyslipidemia in CHD.