Glioblastomas are tumors of neuroectodermal origin with a high degree of diversity. Tumor cells isolated from the surgical material of patients with glioblastomas are heterogeneous populations varying in morphology, phenotype, and genetic characteristics. This paper presents a description of two new glioblastoma cell lines, R1 and T2, isolated from tumor tissue of patients in 2010. We investigated their morphological and cytochemical cell characteristics and the expression of neuronal, mesenchymal, and endothelial markers, as well as the activity of genes encoding a number of growth factors, extracellular matrix proteins, and intracellular proteins typical for cells of mesenchymal origin. R1 and T2 cell lines are morphologically different. T2 cell line was characterized by the presence of multinuclear cells. In terms of the expression of β-tubulin III, MGMT, and p53 protein, R1 cell line was more heterogeneous than T2. R1 and T2 glioblastoma cell lines also differed in the presence and ratio of cell populations with mesenchymal, neuronal, and endothelial markers. Thus, neuronal markers CD133/2 and CD56 were detected only on R1 cells. Both lines were characterized by high activity of growth factor genes TGFβ1, VEGF, and FGF2(b), lower activity of EGF, and high expression of THBS1 and αSMA genes. However, the activity of most of the genes under study in R1 cells was higher than in T2 cells. The greatest difference lay in the expression of HGF, FAP, and TNC. Comparison of two new glioblastoma cell lines, R1 and T2, with the continuously cultivated lines А172 and T98G showed that R1 line had remarkable similarity with A172, while T2 glioblastoma resembled T98G cells. Apparently, the differences between R1 and T2 cell lines are determined by the properties of the initial tumors rather than the time of the cell cultivation.