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Monosomal karyotype (MK) reflects highly unfavorable prognosis in patients with acute myeloid leukemia (AML). This study aimed to study the association of AML-MK with multidrug resistance (MDR) functional activity. A total of 369 AML patients (excluding APL) between 1995 and 2008 at a single center were included retrospectively. Functional MDR activity was evaluated with rhodamine-123 efflux activity with/without verapamil inhibition. MK was noted in 23 patients, only among whom classified into unfavorable cytogenetic risk group. Unfavorable cytogenetic subgroup with MK showed shorter OS (8.7 ± 5.9% vs. 23.5 ± 7.5% at 3 years,P= 0.030), EFS (8.7 ± 5.9% vs. 19.0 ± 6.9% at 3 years,P= 0.029), and a lower CR rate (34.8% vs. 65.7%,P= 0.031) compared with unfavorable subgroup without MK. Functional MDR activity was significantly higher in the unfavorable cytogenetic group with MK compared to all other cytogenetic risk groups taken as a whole (P= 0.026) and showed a trend toward statistical significance when compared with the unfavorable cytogenetic risk group without MK (P= 0.06). AML patients harboring MK showed a poor outcome in terms of lower CR rate and worse EFS/OS, and the presence of MK appeared to be associated with higher MDR functional activity of leukemic blasts.