Objective The human glioblastoma (GBM) U87 cell line is employed as a model for studying the heterogeneity of GBM. This study was to examine the phenotypic profiles and genetic backgrounds of different monoclonal cells derived from the human GBM U87 cell line and explore the molecular mechanisms underlying the phenotypic difference. Methods Using the finite dilution method labeled with 5(6)-carboxyfluorescein diacetate N-hydroxy succinimidyl ester (CFSE), we constructed the monoclonal cell lines CF5 and G11 with typical morphological characteristics derived from the human GBM U87 cell line and identified them by short tandem repeat (STR). We detected the proliferation of the cells by CCK8 assay, EdU incorporation and colony-formation assay, their self-renewal capability by tumor sphere formation assay, their adhesion ability by immunofluorescence and CCK8 adhesion assay, their invasion ability with a 3D culture model, and their sensitivity to chemotherapeutic agents by Annexin V/PI double-staining flow cytometry. We performed transcriptome sequencing and bioinformatics analysis on the genetic profiles and determined the mRNA expressions of the representative differential genes in the enriched pathway by real-time quantitative PCR (qRT-PCR). Results The CF5 and G11 monoclonal cell lines morphologically typical of U87 were successfully constructed, the former small, short and thick, while the latter big, long and thin. Compared with the U87 and G11 cell lines, the CF5 cells showed a significantly higher proliferation ability (P < 0.01), though higher in the U87 than in the G11 cell line, a higher proportion of EdU-positive cells (0.35 ± 0.03 and 0.44 ± 0.03 vs 0.54 ± 0.05, P < 0.01), though higher in the U87 than in the G11 cell line, and a higher tumor-sphere formation ability (P < 0.01), though higher in the U87 than in the G11 cell line. In comparison with the U87 and CF5 cell lines, the G11 cells exhibited remarkably higher abilities of adhesion (P < 0.01) and invasion (P < 0.05), though both higher in the U87 than in the CF5 group. Totally, 159 genes were down-regulated and 303 up-regulated in the CF5 cells compared with those in the U87 and G11 cells, while 281 were down-regulated and 116 up-regulated in the G11 cells compared with those in the CF5 and U87 cells. The CF5 and G11 cells manifested the highest enrichment in the extracellular matrix-associated pathways, which were shown to be closely associated with the invasiveness and drug-resistance of the tumor. Conclusion We successfully constructed human GBM U87-derived monoclonal cell lines CF5 and G11 with different morphological features, phenotypic profiles and genetic backgrounds, which has paved the ground for further studies of the heterogeneity of GBM.