Among lizards, only monitor lizards (Varanidae) have a functionally divided cardiac ventricle. The division results from the concerted function of three partial septa, which may have homology to the ventricular septum of mammals and archosaurs. We show in developing monitors that two septa, the ‘muscular ridge’ and ‘bulbuslamelle’, express the evolutionary conserved transcription factors Tbx5, Irx1 and Irx2, orthologues of which mark the mammalian ventricular septum. Compaction of embryonic trabeculae contributes to the formation of these septa. The septa are positioned, however, to the right of the atrioventricular junction and they do not partake in the separation of incoming atrial blood streams. That separation is accomplished by the ‘vertical septum’ which expresses Tbx3 and Tbx5, which orchestrate the formation of the electrical conduction axis imbedded in the ventricular septum. These expression patterns are more pronounced in monitors than in other lizards, and are associated with a deep electrical activation near the vertical septum, contrasting the primitive base-to-apex activation of other lizards. We conclude that evolutionarily conserved transcriptional programs may underlie the formation of the ventricular septa of monitors.