Chemotherapy resistance is the main reason for treatment failure in acute myeloid leukemia (AML) and the major cause of its mortality. Etoposide is a DNA topoisomerase-II inhibitor that is used either as a single agent or in combination with cytarabine, azacytidine, vinca alkaloids, and anthracyclines for the treatment of relapsed /refractory AML. In this study, we sought to determine and understand the mechanism of etoposide resistance in AML using the HL60 cell line.HL60 cells were treated with incremental doses of etoposide and resistant colonies were isolated by culturing the resistant cells in semi-solid culture media. Three clones were selected for etoposide resistance namely, HL60-EtopR H1A, HL60-EtopR H1B, and HL60-EtopR H1C which demonstrated 4.78, 2.39, and 4.42-fold higher resistance to etoposide compared with the parental cells. To determine molecular differences between the etoposide-resistant HL60-EtopR cells and the parental cells, microarray-based gene expression profiling was performed. We found up regulation of members of the src tyrosine kinase family genes in the etoposide resistant cells. Further studies are required to evaluate the role of Src inhibitors in targeting etoposide resistant cells.