Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.