Background: FLT3/ITD mutations are common in AML and confer a poor prognosis. A sensitive, specific assay for the detection of minimal residual disease (MRD) in FLT3/ITD AML could guide decisions on transplant or maintenance therapy. To date, assays for MRD in FLT3/ITD AML have not been useful due to limited sensitivity. We have developed a sensitive and specific MRD assay for FLT3/ITD mutations using next-generation sequencing (NGS). Methods: Exons 14 and 15 are amplified by PCR and the products were detected by a refined NGS technique developed at Invivoscribe, Inc. Initial validation was carried out by spiking in fixed amounts of mutant DNA into wild type DNA to establish a sensitivity equivalent to detection of at least one ITD-containing cell out of 10,000 with a minimum input of 100,000 cell equivalents of DNA. We validated the assay using marrow from patients with FLT3/ITD AML in CR. The investigator conducting the MRD assay was blind to all clinical and laboratory data. Results: In all samples, the standard CLIA-certified assay for the FLT3/ITD mutation was negative and no leukemia was evident by morphology. In 4 samples from patients in first CR prior to consolidation, the mutation was detected with levels ranging from 1.35E-05 to 1.74E-04 mutant ITD reads/total reads. In 3 patients who had relapsed and had achieved CR2, the mutation was detected at levels ranging from 1.38E-06 to 1.11E-04. The next 6 samples were collected during post-transplant surveillance from patients who had undergone transplant in CR. No mutation was detected in any of these patients, all 6 of whom are alive and disease free 2.5-5.5 years after transplant. Finally, in 2 samples from patients transplanted in CR1 (both of whom were in morphologic CR with 100% donor chimerism in marrow and T-cells), the MRD assay detected the mutations at levels of 3.67E-03 and 1.04E-04, respectively. Both patients relapsed with FLT3/ITD AML within 6 months of these specimens being collected. Conclusions: This novel MRD assay is specific, and is 2 orders of magnitude more sensitive than current commercially available assays for FLT3/ITD mutations. We anticipate that this assay will be broadly available to the public soon, and will have a significant impact in the clinical management of this disease.