Abstract Subclonal mutations, that are present in a small subset of tumor cells prior to chemotherapy, could serve as a reservoir for the emergence of drug resistance. These could selectively expand during chemotherapy and result in relapse and death. We have used Duplex DNA Sequencing (DS) to detect low frequency subclonal mutations in AML, both prior to treatment and after relapse. DS focuses on a limited number of target genes at the level of single DNA molecules. Mutations are scored only if they are present at the same position in both strands of the same DNA molecule and are complementary, resulting in sequencing accuracy that is more than 10,000-fold greater than that of conventional next-generation DNA sequencing (NGS). To follow the course of mutation accumulation in AML, we used capture hybridization to enrich for 15 genes previously reported to be mutated in AML. We identified multiple subclonal mutations prior to therapy that would not be detected by routine NGS. For example, in one relapse sample, we identified a mutation in NRAS that was present in 32% of the cells. The identical mutation was detected by DS prior to treatment in 1% of cells. To further assess the frequency of subclonal mutations in AML, we targeted a 30 kb panel of genes at 500,000 fold depth, which facilitates detection of mutations present in fewer than 0.01% of cells. 5 subclonal mutations were identified within the 30 kb target, corresponding to a frequency of 170 mutations per megabase, Extrapolation of these results to the whole genome indicates that there are approximately 500,000 subclonal mutations per AML genome, which is 1,000 fold higher than the frequency of clonal mutations. These subclonal mutations would be unevenly distributed throughout the genome and be an important source for the selection of drug resistance during therapy. Citation Format: Lawrence A. Loeb, Michael W. Schmitt, Mark J. Prindle, Pamela S. Becker. Duplex sequencing of AML reveals extensive sub clonal heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4890. doi:10.1158/1538-7445.AM2015-4890