Acute myeloid leukemia (AML) is a heterogeneous group of disorders characterized by abnormal proliferation of myeloid precursors and a maturation block. Underlying genetic lesions determine an altered expression program (transcriptosome) that can be studied in depth by massive technologies. Alternatively, we selected a pathway profiling strategy based on the current knowledge in order to stratify de novo AML patients and identify those cases which would potentially benefit from the use of new chemotherapeutic agents. One hundred and thirty-two RNA samples obtained from de novo adult AML patients were tested for FLT3, FLT3-LG, NDST1, HDAC2, ATRX, FOS, DNMT1, DNMT3A, DNMT3B, NBS1, RAD50, MRE11A, Meis1 and Meis2 expression using quantitative PCR (qPCR) assays. Clinical and biologic findings were correlated with expression results. Cluster analysis was also performed. FLT3 expression defined three subgroups of patients. The best outcome was found in the group with the lowest FLT3 expression. Intermediate levels of FLT3 were associated with the worst outcome. Patients with low levels of ATRX more frequently presented an adverse karyotype whereas cases with preserved ATRX levels showed an excellent outcome. In accordance with previous results, Meis1 downregulation is a useful surrogate marker indicating a good prognosis in AML patients. Simple qPCR platforms may help to identify different biologic subgroups in AML.