The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiac lineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5−, TBX5−NKX2-5+, and TBX5−NKX2-5−, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate the investigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling. Display omitted •TBX5Clover2 and NKX2-5TagRFP reporter enables purification of 4 cardiac subpopulations•Different cardiac lineages differentiate into specific cardiac cell types•CORIN is a cell-surface marker for the TBX5+NKX2-5+ subpopulation•Lineage-specific cardiomyocyte subtypes can be used for precise drug testing Wu and colleagues create a double-knockin human TBX5Clover2 and NKX2-5TagRFP reporter to delineate cardiac lineages during differentiation. Molecular and functional analyses uncover that differential expression of TBX5 and NKX2-5 specifies different cardiac fate. This reporter is applicable for studying cardiac lineage determination in vitro and isolating cardiac subpopulations.