Background: Genome-wide association studies have identified hundreds of loci that are robustly associated with circulating lipids, atherosclerosis and coronary artery disease (CAD). In all but a few of these loci the causal genes and mechanisms remain unknown. Small-scale studies suggest that zebrafish larvae represent a promising model system for genetic screens in dyslipidemia, early-stage atherosclerosis and CAD. We expanded the phenotypic pipeline, increased the throughput, and performed a large-scale validation study. Methods: We used a three-tiered approach to validate the zebrafish model system: 1) a dietary intervention to examine the effect of overfeeding and cholesterol supplementation (N=1956); 2) treatment with atorvastatin and ezetimibe (N=835); and 3) a genetic screen for zebrafish orthologues of APOE, APOB and LDLR using a multiplex CRISPR-Cas9 approach (N=330). After imaging of vascular atherogenic traits and body size, whole-body lipid and glucose levels were assessed using enzymatic assays, and CRISPR-Cas9 target sites were sequenced on a MiSeq. Results: Overfeeding and cholesterol supplementation have independent pro-atherogenic effects, including higher LDLc and triglyceride levels, more vascular infiltration by lipids and oxidized LDLc, and more vascular co-localization of lipids with macrophages and neutrophils. Treatment with atorvastatin and ezetimibe can largely prevent this diet-induced pro-atherogenic state, but results in higher glucose levels. Finally, mutations in apoea and apoeb result in higher whole-body LDLc levels; mutations in apobb.1 result in more vascular co-localization of lipids and neutrophils; and mutations in ldlra result in more vascular infiltration by lipids, and in more vascular co-localization of lipids and macrophages. Data from all larvae combined show that atherosclerosis in 10-day-old zebrafish larvae is mainly driven by higher triglyceride levels. Conclusion: Systematic genetic screens in zebrafish larvae will likely help increase our understanding of disease aetiology and identify new targets that can be translated into efficient medication for prevention and treatment.