Aim: To characterize and compare the different biological behaviors of 2 novel human osteosarcoma cell lines, Zos and Zos‐M, established respectively from the primary tumor and the skip metastasis of an osteosarcoma patient. Methods:In vitro studies included morphological observations, karyotype analysis, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide cell proliferation assay, and cell sensitivity to chemotherapeutic drugs. Subcutaneous and intravenous inoculations into nude mice were carried out to study the tumorigenicity and the metastatic potential. RT‐PCR was performed to assess the expression of the osteoblastic markers and some metastasis‐related genes. Results: Both cell lines remained stable for more than 100 passages in vitro without interruption. The RT‐PCR examination indicated that they retained the molecular characteristics of an osteoblastic lineage. The karyotype analysis displayed aneuploidy and various structural abnormalities. Both cell lines are tumorigenic; Zos‐M differs from Zos by the former's ability to develop lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis‐related genes revealed that the decreased expression of cadherin‐11 in Zos‐M may correlate with a high potential of metastases. Moreover, both cell lines are less sensitive to the current chemotherapy protocols. Conclusion: The establishment of osteosarcoma cell lines, Zos and Zos‐M, and related animal models provide a useful resource for studying the aggressive behavior of osteosarcoma and will be helpful for screening effective treatment strategies.