Drug resistance is often a limiting factor in successful chemotherapy. Understanding of the molecular mechanism of resistance to methotrexate is important for optimal use as well as for the development of new drugs. Using standard cytogenetic techniques, double minutes (DMs) were detected in mouse methotrexate-resistant cell lines inducted in vitro in this study. Moreover, functional SuperArray, determining the regulation of gene expression corresponding to 96 genes involved in the metabolism of and the development of resistance to cancer drugs was used to study in detail the molecular mechanism of resistance to methotrexate. Significant higher expressions of many genes especially dhfr, mrp5, atm and p53 were detected in methotrexate-resistant cells than in normal mouse cells by SuperArray analysis and corresponding excessive expressions of DHFR, MRP5, ATM, and P53 proteins in methotrexate-resistant cells were also confirmed by Western blot analysis. The results in this study indicated that DHFR, MRP5, ATM, and P53 could play important roles in resistance to methotrexate and some of them would be new potential drug targets.