Cardiac gene transfer is an attractive tool for developing novel heart disease treatments. Adeno-associated viral (AAV) vectors are widely used to mediate transgene expression in animal models and are being evaluated for human gene therapy. However, it is not clear which serotype displays the best cardiac tropism. Therefore, we curried out this study to directly compare AAV serotypes 1–9 heart transduction efficiency after indirect intracoronary injection. AAV-cytomegalovirus immediate early enhancer promoter (CMV)-luciferase serotypes 1–9 were injected in the left ventricular cavity of adult mice, after cross-clamping the ascending aorta and pulmonary artery. An imaging system was used to visualize luciferase expression at 3, 7, 21, 70, and 140 days postinjection. Echocardiography was performed to evaluate cardiac function on day 140. At the end of the study, luciferase enzyme activity and genome copies of the different AAV serotypes were assessed in several tissues and potential AAV immunogenicity was evaluated on heart sections by staining for macrophage and lymphocyte antigens. Among AAV serotypes 1–9, AAV6 showed the best capability of achieving high transduction levels in the myocardium in a tissue-specific manner, whereas the other serotypes had less cardiac transduction and more extracardiac expression, especially in the liver. Importantly, none of the serotypes tested with this marker gene affected cardiac function nor was associated with inflammation.