Mammalian heart development involves complex morphogenetic events which lead to the formation of fully separated left and right atrial and ventricular chambers from a tubular heart. Separation of left and right ventricular chambers is dependent on a single structure, the interventricular septum (IVS), which has both muscular and mesenchymal components. Little is known about the morphogenetic events that lead to the formation of the muscular component of the IVS. We have analyzed two transgenic mouse lines that display complementary nlacZ reporter gene expression patterns in the embryonic ventricles: the Mlc1v-nlacZ-24 transgene is expressed in right ventricular myocardium and the Mlc3f-nlacZ-2 transgene in left ventricular myocardium. Detailed analysis of these transgene expression patterns during IVS formation reveals a symmetric left and right myocardial identity within the developing IVS between embryonic days 9.5 and 11.5. From embryonic day 12.5 onwards, myocytes with a left ventricular identity dominate the IVS, particularly in its dorsal aspect. The T-box transcription factor encoding gene, Tbx18, is expressed in the left ventricle and left side of the developing IVS, providing additional support for the presence of left and right ventricular identities within the IVS. Analysis of clonally related cardiomyocyte clusters confirms that both left and right ventricular myocardial cell populations contribute to the forming IVS, in similar domains to those defined by the Mlc-nlacZ transgenes. Examination of the orientation as well as the distribution of labeled cells in clusters provides new insights into the morphogenesis of the septum.