In vertebrates, the heart has two main layers of cardiac muscle, a peripheral compact layer and an internal trabecular layer. Little is known on the differerences in gene expression between both layers. In zebrafish the outer layer is named cortical layer and the internal also trabecular layer. Here we used a double transgenic line labelling with GFP tbx5-positive cells and cardiomyoctes with nuclear DsRed (nucDsRed) to distinguish cortical from trabecular myocardium. Then, we compared the transcriptome of trabecular and cortical myocardium in the adult zebrafish. We describe that Tbx5a is a good marker of trabecular myocardium. Overall design: Four paired biological replicates consisting on Tbx5-positive and Tbx5-negative adult zebrafish ventricular cardiomyocytes were analysed by RNA-seq to compare their transcriptomic profiles.