To better understand the molecular mechanisms of multidrug resistance (MDR) of human cancers, we isolated differentially expressed genes from drug-resistant human gastric adenocarcinoma cell lines using a polymerase chain reaction-based subtractive hybridization technique. Sixty three genes were identified to be differentially expressed in the drug-resistant human gastric adenocarcinoma cell lines. Among the 63 up-regulated genes, 27 were known genes, of which two have been reported to be associated with MDR. The remaining ones were undefined genes or expressed sequenced tags. The results suggest that this strategy is efficient for large-scale cloning of differentially expressed genes in drug-resistant cells. Further characterization of these genes will shed more light on the understanding of molecular mechanisms of MDR of human cancer cells.