Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K-d) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas.