Purpose: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical development as a cancer therapeutic agent. Many human malignant glioma cells, however, are resistant to TRAIL treatment. We, therefore, investigated the genomic alterations in TRAIL-resistant malignant glioma cells. Experimental Design: Seven glioma cell lines and two primary cultures were first analyzed for their sensitivity to TRAIL and chemotherapy and then examined for the genomic alterations in key TRAIL apoptotic genes by comparative genomic hybridization (CGH), G-banding/spectral karyotyping, and fluorescence in situ hybridization (FISH). Results: CGH detected loss of the chromosomal regions that contain the following genes: 8p12-p23 (DR4 and DR5), 2q33-34 (caspase-8), 11q13.3 (FADD), 22q11.2 (Bid), and 12q24.1-q24.3 (Smac/DIABLO) in TRAIL-resistant cell lines. Spectral karyotyping showed numerical and structural aberrations involving the chromosomal regions harboring these genes. A combination of G-banding/spectral karyotyping and FISH further defined the loss or gain of gene copy of these genes and further showed the simultaneous loss of one copy of DR4/DR5, caspase-8, Bid, and Smac in two near-triploid cell lines that were resistant to the combination treatment with TRAIL and chemotherapy. Loss of the caspase-8 locus was also detected in a primary culture in correlation with the culture resistance to the combined TRAIL and chemotherapy treatment. Conclusions: The study identifies chromosomal alterations in TRAIL apoptotic genes in the glioma cells that are resistant to the treatment with TRAIL and chemotherapy. These genetic alterations could be used to predict the responsiveness of malignant gliomas to TRAIL-based therapies in clinical treatment of the tumors. © 2006 American Association for Cancer Research.