Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression ofHoxb1in the anterior SHF results in hypoplastic right ventricle. Activation ofHoxb1in embryonic stem cells arrests cardiac differentiation, whereasHoxb1-deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking bothHoxb1and its paralogHoxa1results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.