Accumulating evidence supports that cancer stem cells (CSCs) are responsible for tumor initiation, progression, distal metastasis and even drug resistance. Although CD90 has been identified as a marker for several types of stem cells, such as liver CSCs, the potential role of CD90 as a marker for lung CSCs has yet to be fully characterized. Our previous study demonstrated that the lung cancer stem-like cells isolated from A549 tumor spheres, which were cultured in serum-free conditioned medium, had stronger proliferation and self-renewal abilities, and expressed higher levels of the stem cell markers Sox2 and Oct4 as compared to A549 adherent cells. In the present study, we identified CD90 as a novel surface marker of CSCs in lung cancer cells. Furthermore, we isolated CD90+ CSCs from lung cancer cell lines A549 and H446. Our results revealed that the CD90+ cells, but not the CD90- cells, from lung cancer cells displayed higher tumorigenic capacity. These findings suggest that CD90 could be a potential marker of lung CSCs and thus provide new insight into further therapeutic strategies of lung cancer.