Abstract Body: The sinoatrial node (SAN) consists of distinct anatomical compartments that each contain specialized pacemaker cardiomyocytes, which have distinct gene expression profiles and functional roles in cardiac pacemaking. The SAN head, located cranially, is densely innervated and more active when sympathetic tone is high; while the SAN tail located more caudally, is more active when parasympathetic tone is high. Finally, a transitional region regulates impulse transition to the atrial myocardium. The timing, lineage dynamics, and molecular cues that control the emergence and diversification of SAN progenitors in the mammalian embryo are incompletely understood. Using comparative ATAC-seq data on neonatal pacemaker and right atrium cardiomyocytes, our laboratory previously identified a differentially accessible chromatin region corresponding to an evolutionarily conserved enhancer of the transcription factor Isl1 in the developing SAN (Isl1-SAN-Enhancer, ISE). To gain insight into the lineage dynamics of SAN progenitors, we crossed ISE-hsp68-CreERT2 transgenic mice with a Rosa^ZsGreen reporter line. Early-stage tamoxifen-induced Cre recombinase activation between E7.5 and E12.5 revealed initial broad labeling of the cardiac myocardium and some non-myocyte lineages, with narrowing of fate to the SAN and dorsal right atrium after E8.5. By E9.5, ISE fate became increasingly restricted to the SAN head, demonstrating greater specificity for SAN labeling than Hcn4^CreERT2 or Isl1^CreEsr1 knock-in mouse models. Clonal fate mapping using Mosaic Analysis with Double Markers (MADM) system allowed the fate mapping of single ISE+ clones. Tamoxifen-induction of ISE-CreERT2; MADM^GT/TG embryos between E7.5 and E8.5 and immunohistochemical co-staining for key pacemaker markers, including Hcn4 and Nkx2.5, showed that the majority of twin spots were confined to one cell type rather than spanning multiple compartments. This evidence suggests a model of early lineage segregation among different SAN pacemaker cardiomyocyte subtypes (SAN head and non-SAN head). Ongoing single cell multi-omics analysis from ISE+ progenitors and their progenies dissected at E8.25, E8.5, E8.75, and E9.25 aim to identify the transcriptional and epigenetic regulators that drive early commitment to different pacemaker lineages. Knowledge of the temporal and molecular drivers of SAN fate commitment may inform regenerative approaches for pacemaker cell therapy.