Since its use in the initial characterization of an acute myeloid leukemia (AML) genome, next-generation sequencing (NGS) has continued to molecularly refine the disease. Here, the authors review the spectrum of NGS applications that have subsequently delineated the prognostic significance and biologic consequences of these mutations. Furthermore, the role of this technology in providing a high-resolution glimpse of AML clonal heterogeneity, which may inform future choice of targeted therapy, is discussed. Although obstacles remain in applying these techniques clinically, they have already had an impact on patient care. Cancer 2014;120:1134–1144. © 2014 American Cancer Society.