Successful clinical remission to therapy for acute myeloid leukemia (AML) is required for long-term survival to be achieved. Despite trends in improved survival due to better supportive care, up to 40% of patients will have refractory disease, which has a poorly understood biology and carries a dismal prognosis. The development of effective treatment strategies has been hindered by a general lack of knowledge about mechanisms of chemotherapy resistance. Here, through transcriptomic analysis of 154 cases of treatment-naive AML, three chemorefractory patient groups with distinct expression profiles are identified. A classifier, four key refractory gene signatures (RG4), trained based on the expression profile of the highest risk refractory patients, validated in an independent cohort (n = 131), was prognostic for overall survival (OS) and refined an established 17-gene stemness score. Refractory subpopulations have differential expression in pathways involved in cell cycle, transcription, translation, metabolism, and/or stem cell properties. Ex vivo drug sensitivity to 122 small-molecule inhibitors revealed effective group-specific targeting of pathways among these three refractory groups. Gene expression profiling by RNA sequencing had a suboptimal ability to correctly predict those individuals resistant to conventional cytotoxic induction therapy, but could risk-stratify for OS and identify subjects most likely to have superior responses to a specific alternative therapy. Such personalized therapy may be studied prospectively in clinical trials.