A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV) in data from 53,174 individuals. However, functional follow-up experiments - aiming to identify and characterize causal genes in these loci - have not yet been performed. We developed an image- and CRISPR-Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos and larvae. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in fertilized eggs from fish that transgenically express GFP on smooth muscle cells (Tg( acta2:GFP )), to visualize the beating heart using a fluorescence microscope. An automated analysis of repeated 30s recordings of 381 live zebrafish atria at 2 and 5 days post-fertilization highlighted genes that influence HRV ( hcn4 and si:dkey-65j6.2 ); heart rate ( rgs6 and hcn4 ) and the risk of sinoatrial pauses and arrests ( hcn4 ). Hence, our screen confirmed the role of established genes for heart rate and rhythm ( rgs6 and hcn4 ), and highlighted a novel gene implicated in HRV ( si:dkey-65j6.2 ).