Epidermal growth factor receptor (EGFR) overexpression occurs in nearly 50% of cases of glioblastoma (GBM), but its clinical and biological implications are not well understood. We have used Affymetrix high-density oligonucleotide arrays to demonstrate that EGFR-overexpressing GBMs (EGFR+) have a distinct global gene transcriptional profile. We show that the expression of 90 genes can distinguish EGFR+ from EGFR nonexpressing (EGFR−) GBMs, including a number of genes known to act as growth/survival factors for GBMs. We have also uncovered two additional novel molecular subtypes of GBMs, one of which is characterized by coordinate upregulation of contiguous genes on chromosome 12q13–15 and expression of both astrocytic and oligodendroglial genes. These results define distinct molecular subtypes of GBMs that may be important in disease stratification, and in the discovery and assessment of GBM treatment strategies.