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Rationale: Genetic testing for Long QT Syndrome is now a standard and integral component of clinical cardiology. A major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants in a high-throughput manner. Objective: The goal of this study was to design and test a high-throughput in vivo cardiac assay to distinguish between disease-causing and benign KCNH2 ( hERG1) variants, using the zebrafish as a model organism. Methods and Results: We tested the ability of previously characterized Long QT Syndrome hERG1 mutations and polymorphisms to restore normal repolarization in the kcnh2 -knockdown embryonic zebrafish. The cardiac assay correctly identified a benign variant in 9 of 10 cases (negative predictive value 90%), whereas correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%). Conclusions: The in vivo zebrafish cardiac assay approaches the accuracy of the current benchmark in vitro assay for the detection of disease-causing mutations, and is far superior in terms of throughput rate. Together with emerging algorithms for interpreting a positive long QT syndrome genetic test, the zebrafish cardiac assay provides an additional tool for the final determination of pathogenicity of gene variants identified in long QT syndrome genetic screening.