This study identifies a subpopulation of human malignant melanoma cells, defined by the expression of the chemoresistance mediator ABCB5, that is enriched for tumor-initiating cells (TICs). These ABCB5+ TICs exhibit a primitive molecular phenotype and correlate with clinical melanoma progression. In xenotransplantation experiments, ABCB5+ melanoma cells show greater tumorigenic capacity than ABCB5- cells and re-establish clinical tumor heterogeneity. Genetic lineage tracking demonstrates that ABCB5+ TICs have the capacity for self-renewal and differentiation, generating both ABCB5+ and ABCB5- progeny. Systemic administration of a monoclonal antibody targeting ABCB5 inhibits tumor growth, suggesting that specific targeting of this TIC subset could be a strategy to eradicate cancers resistant to systemic therapy. The findings highlight the potential of ABCB5 as a universal marker for TICs and a therapeutic target in advanced malignant melanoma.This study identifies a subpopulation of human malignant melanoma cells, defined by the expression of the chemoresistance mediator ABCB5, that is enriched for tumor-initiating cells (TICs). These ABCB5+ TICs exhibit a primitive molecular phenotype and correlate with clinical melanoma progression. In xenotransplantation experiments, ABCB5+ melanoma cells show greater tumorigenic capacity than ABCB5- cells and re-establish clinical tumor heterogeneity. Genetic lineage tracking demonstrates that ABCB5+ TICs have the capacity for self-renewal and differentiation, generating both ABCB5+ and ABCB5- progeny. Systemic administration of a monoclonal antibody targeting ABCB5 inhibits tumor growth, suggesting that specific targeting of this TIC subset could be a strategy to eradicate cancers resistant to systemic therapy. The findings highlight the potential of ABCB5 as a universal marker for TICs and a therapeutic target in advanced malignant melanoma.