Background: Multidrug resistance represents a major obstacle in the chemotherapeutic treatment of acute leukemia (AL). Adenosine triphosphate ATP-binding Cassette (ABCB5) and Multidrug resistance (MDR1) genes are integral membrane proteins belonging to ATP-binding cassette transporters superfamily. Purpose: The present work aimed to investigate the impact of ABCB5 and MDR1 genes expression on the response to chemotherapy in a cohort of Egyptian acute leukemia patients. The study included 90 patients; 53 AML cases and 37 ALL cases in addition to 20 healthy volunteers as controls. Methods: Quantitative assessment of MDR1 and ABCB5 genes expression was performed by quantitative real-time polymerase chain reaction (qPCR). Additional prognostic molecular markers were determined as internal tandem duplications of the FLT3 gene (FLT3-ITD) and Nucleophosmin gene mutation (NMP1) for AML cases, and mbcr-abl fusion transcript for B-ALL cases. Results: In AML patients, ABCB5 and MDR1 expression levels did not differ significantly between de novo or relapsed cases and did not correlate with the overall survival (OS) or disease free survival (DFS). AML patients were stratified according to the studied genetic markers and complete remission rate was found to be more prominent in patients having negative expression of MDR1 and ABCB5 genes together with mutated NPM1 gene. In ALL patients, ABCB5 gene expression level was significantly higher in relapsed cases and MDR1 gene expression was significantly higher in patients with resistant disease. Conclusion: In conclusion, the results obtained by the current study provide additional evidence of the role played by these genes as predictive factors for resistance of leukemic cells to chemotherapy and hence treatment outcome.