Tumor cell lines are an indispensable tool for cancer research. However, among cell lines of the same pathological group, heterogeneity has been detected in gene expression, gene mutation, and cellular response to various treatments. In this study, we systematically investigated the extent of heterogeneity of gene expression in three glioblastoma cell lines using cDNA array technology in which the expression of 588 cellular genes is studied simultaneously. Comparison of the expression profiles revealed substantial qualitative and quantitative heterogeneity. Among the 588 genes, 197 genes were expressed in all three lines and 56 genes were not expressed in any of the three lines; total of 222 genes were expressed in only two of the three cell lines, and 113 genes were expressed in only one of the three cell lines. These results provide molecular evidence that cell lines of the same pathological origin can be highly heterogeneous.