The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3+ pacemaker cells. Here we isolated and characterized the Tbx3+ cells from Tbx3+/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3+ pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3+ sinoatrial nodal cells and Nppb-Katushka+ atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program including key nodal transcription factors, BMP-signaling, and Smoc2, which disruption in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1, HOX family members, and BMP- and NOTCH-signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3+/Venus allele provides a reliable tool to visualize the sinoatrial node and to study its development and function.