The identification of breast tumor initiating cells (BTICs) is important for the diagnosis and therapy of breast cancers. This study was undertaken to evaluate whether the extra domain-B of fibronectin (EDB-FN) could be used as a new biomarker for BTICs and whether EDB-FN targeting superparamagnetic iron oxide nanoparticles (SPIONs) could be used as a magnetic resonance imaging (MRI) contrast agent for BTIC imaging in vitro and in vivo. BTICs (NDY-1) exhibited high EDB-FN expression, whereas non-BTICs (MCF-7, BT-474, SUM-225, MDA-MB-231) did not exhibit EDB-FN expression. Furthermore, Cy3.3-labeled EDB-FN specific peptides (APT(EDB)) showed preferential binding to the targeted NDY-1 cells. To construct an EDB-FN targeted imaging probe, APT(EDB) was covalently attached to a thermally cross-linked SPION (TCL-SPION) to yield APT(EDB)-TCL-SPION. In the in vitro MRI of cell phantoms, selective binding of APT(EDB)-TCL-SPION to NDY-1 cells was evident, but little binding was observed in MCF-7 cells. After the intravenous injection of APT(EDB)-TCL-SPION into the NDY-1 mouse tumor xenograft model, a significant decrease in the signal within the tumor was observed in the T-2*-weighted images; however, there was only a marginal change in the signal of non-targeting SPIONs such as APT(scramble)-TCL-SPION or TCL-SPION. Taken together, we report for the first time that EDB-FN was abundantly expressed in BTICs and may therefore be useful as a new biomarker for identifying BTICs. Our study also suggests that APT(EDB)-TCL-SPION could be used as an MRI contrast agent for BTIC imaging.