Primary breast sarcomas (PBSs) that arise from mammary stroma are very rare, highly aggressive and therapy- resistant tumors with a heterogeneous phenotype. In this study, we sought to identify tumor-initiating cells (TICs) in PBSs and to describe their features. We isolated long-term self-renewing sarcospheres (designated NDY-1) from primary breast carcinosarcoma tissue (sarcoma component >95%) using the anchorage-independent culture method. NDY-1 spheres expressed various mesenchymal cell markers, and their tumorigenic potential was markedly reduced in adherent culture conditions, compared to spheres. Screening for integrins revealed a marked decrease in CD49d expression in adherent culture conditions of NDY-1. The CD49d+/high subpopulation sorted from NDY-1 spheres displayed higher cell viability and sphere-forming ability than CD49d-/low population in vitro. Moreover, the CD49d+/high population displayed high tumor initiating ability in limiting dilution transplantation to NOD/SCID mice, and the xenotransplanted CD49d+/high population recapitulated the complexity of the original primary tumors. Greater doxorubicin resistance was exhibited by the CD49d+/high population, compared with the CD49d-/low population. Thus, our results collectively demonstrate that CD49d+/high cells from sarcospheres display enhanced sphere-forming, drug resistance and tumor-initiating abilities. To our knowledge, this is the first study to identify TICs from breast sarcoma.