Importance:Mitochondrial dysfunction is a core component of the aging process and may play a key role in atherosclerotic cardiovascular disease. Mitochondrial DNA copy number (mtDNA-CN), which represents the number of mitochondria per cell and number of mitochondrial genomes per mitochondrion, is an indirect biomarker of mitochondrial function. Objective:To determine whether mtDNA-CN, measured in an easily accessible tissue (buffy coat/circulating leukocytes), can improve risk classification for cardiovascular disease (CVD) and help guide initiation of statin therapy for primary prevention of CVD. Design, Setting, and Participants:Prospective, population-based cohort analysis including 21 870 participants (20 163 free from CVD at baseline) from 3 studies: Cardiovascular Health Study (CHS), Atherosclerosis Risk in Communities Study (ARIC), and Multiethnic Study of Atherosclerosis (MESA). The mean follow-up was 13.5 years. The study included 11 153 participants from ARIC, 4830 from CHS, and 5887 from MESA. Analysis of the data was conducted from March 10, 2014, to January 29, 2017. Exposures:Mitochondrial DNA-CN measured from buffy coat/circulating leukocytes. Main Outcomes and Measures:Incident CVD, which combines coronary heart disease, defined as the first incident myocardial infarction or death owing to coronary heart disease, and stroke, defined as the first nonfatal stroke or death owing to stroke. Results:Of the 21 870 participants, the mean age was 62.4 years (ARIC, 57.9 years; MESA, 62.4 years; and CHS, 72.5 years), and 54.7% of participants were women. The hazard ratios for incident coronary heart disease, stroke, and CVD associated with a 1-SD decrease in mtDNA-CN were 1.29 (95% CI, 1.24-1.33), 1.11 (95% CI, 1.06-1.16), and 1.23 (95% CI, 1.19-1.26). The associations persisted after adjustment for traditional CVD risk factors. Addition of mtDNA-CN to the 2013 American College of Cardiology/American Heart Association Pooled Cohorts Equations for estimating 10-year hard atherosclerosis CVD risk was associated with improved risk classification (continuous net reclassification index, 0.194; 95% CI, 0.130-0.258; P < .001). Mitochondrial DNA-CN further improved sensitivity and specificity for the 2013 American College of Cardiology/American Heart Association recommendations on initiating statin therapy for primary prevention of ASCVD (net 221 individuals appropriately downclassified and net 15 individuals appropriately upclassified). Conclusions and Relevance:Mitochondrial DNA-CN was independently associated with incident CVD in 3 large prospective studies and may have potential clinical utility in improving CVD risk classification.