Glioblastomas are invasive and aggressive tumors of the brain, generally considered to arise from glial cells. A subset of these cancers develops from lower-grade gliomas and can thus be clinically classified as "secondary," whereas some glioblastomas occur with no prior evidence of a lower-grade tumor and can be clinically classified as "primary." Substantial genetic differences between these groups of glioblastomas have been identified previously. We used large-scale expression analyses to identify glioblastoma-associated genes (GAG) that are associated with a more malignant phenotype via comparison with lower-grade astrocytomas. We have further defined gene expression differences that distinguish primary and secondary glioblastomas. GAGs distinct to primary or secondary tumors provided information on the heterogeneous properties and apparently distinct oncogenic mechanisms of these tumors. Secondary GAGs primarily include mitotic cell cycle components, suggesting the loss of function in prominent cell cycle regulators, whereas primary GAGs highlight genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences. These data highlight that the development of gene pathway-targeted therapies may need to be specifically tailored to each subtype of glioblastoma.