Gliomas are the most common primary brain tumors. The most malignant form, the glioblastoma multiforme (GBM; WHO IV), is characterized by an invasive phenotype, which enables the tumor cells to infiltrate into adjacent brain tissue. When investigating GBM migration and invasion properties in vitro, in most cases GBM cell lines were analyzed. Comprehensive investigations focusing on progression-dependent characteristics of migration processes using fresh human glioma samples of different malignancy grades do not exist. Thus, we isolated fast-migrating tumor cells from fresh human glioma samples of different malignancy grades (astrocytomas WHO grade II, grade III, GBM, and GBM recurrences) and characterized them with regard to the transcription of genes involved in the migration and invasion, tumor progression, epithelial-to-mesenchymal transition, and stemness. In addition, we transferred our results to GBM cell lines and glioma stem-like cells and examined the influence of temozolomide on the expression of the above-mentioned genes in relation to migratory potential. Our results indicate that "evolutionary-like" expression alterations occur during glioma progression when comparing slow- and fast-migrating cells of fresh human gliomas. Furthermore, a close relation between migratory and stemness properties seems to be most likely. Variations in gene expression were also identified in GBM cell lines, not only when comparing fast- and slow-migrating cells but also regarding temozolomide-treated and untreated cells. Moreover, these differences coincided with the expression of stem cell markers and their migratory potential. Expression of migration-related genes in fast-migrating glioma cells is not only regulated in a progression-dependent manner, but these cells are also characterized by specific stem cell-like features.