Abstract: Cardiomyopathy is a common disease and is regarded as an important cause of premature death. The development of a gene therapy vector for cardiomyopathy is the key to gene therapy of cardiomyopathy. In order to build a gene therapy vector targeting myocardium, the eight selected AAV (AAV1−AAV4, AAV6−AAV9) capsid genes were recombined by DNA shuffling technology, and a random mutant AAV capsid library was constructed in mice direct screening. Finally the new AAV capsids with high myocardial targeting and low liver targeting, AAVH50 and AAVH59, were screened. Through the tail vein injection, the luciferase (Luc) gene carried was delivered to mice. It was found that among the eleven tissues tested, the gene expressions of AAVH50 and AAVH59 in myocardial tissue were higher than other tissues. Compared with AAV6 and AAV9, AAVH50 and AAVH59 expressed similar levels of luciferase in the myocardium, and the expression in the liver significantly decreased AAV9. After AAVH50 and AAVH59 infected primary neonatal rat cardiomyocytes, the efficiency of the selected AAVH59 in direct myocardial infection was significantly higher than that of AAV9. Therefore, this study screened to obtain a new type of AAV vector with high targeting to myocardium, and to the liver and low targeting, which is expected to provide a new type of AAV vector for gene therapy of myocardial diseases.