Adeno-associated virus serotype 9 (AAV9) is often used in heart research involving gene delivery due to its cardiotropism, high transduction efficiency, and little to no pathogenicity, making it highly applicable for gene manipulation, in vivo. However, current AAV9 technology is limited by the lack of strains that can selectively express and elucidate gene function in an atrial- and ventricular-specific manner. In fact, study of gene function in cardiac atria has been limited due to the lack of an appropriate tool to study atrial gene expression in vivo, hindering progress in the study of atrial-specific diseases such as atrial fibrillation, the most common cardiac arrhythmia in the USA. This chapter describes the method for the design and production of such chamber-specific AAV9 vectors, with the use of Nppa and Myl2 promoters to enhance atrial- and ventricular-specific expression. While several gene promoter candidates were considered and tested, Nppa and Myl2 were selected for use here because of their clearly defined regulatory elements that confer cardiac chamber-specific expression. Accordingly, Nppa (−425/+25) and Myl2 (−226/+36) promoter fragments are inserted into AAV9 vectors. The atrial- and ventricular-specific expression conferred by these new recombinant AAV9 was confirmed in a double-fluorescent Cre-dependent reporter mouse model. At only 450 and 262 base pairs of Nppa and Myl2 promoters, respectively, these AAV9 that drive chamber-specific AAV9 transgene expression address two major limitations of AAV9 technology, i.e., achieving chamber-specificity while maximizing space in the AAV genome for insertion of larger transgenes.