Background Cardiac gene therapy using the adeno‐associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off‐target localization. To overcome this, studies have applied cardiac‐specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial‐specific gene expression, an enhancer calsequestrin cis‐ regulatory module 4 ( CRM4 ) and the highly atrial‐specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. Methods To observe expression and bio‐distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin ( CRM4 .SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. Results The CMV promoter containing vectors showed the strongest expression in vitro and in vivo . However, the module SLN combination showed enhanced atrial expression and a minimized off‐target effect even when compared with the individual SLN promoter. Conclusions For gene therapy involving atrial gene transfer, the CRM4 .SLN combination is a promising alternative to the use of the CMV promoter. CRM4 .SLN had significant atrial expression and minimized extra‐atrial expression.