Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in non-dividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2. Imaging-based sequential evaluation of endogenously tagged protein revealed that HDR occurs in cardiomyocytes, independently of DNA synthesis. We sought to repair a pathological mutation in Tnnt2 in cardiomyocytes of cardiomyopathy model mice. An sgRNA that avoided the mutated exon minimized deleterious effects on Tnnt2 expression, and AAV-mediated HDR achieved precise genome correction at a frequency of ~12.5%. Thus, targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.