Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) both represent highly heterogeneous entities on the basis of diverse cyto- and molecular genetic alterations with considerable influence on prognosis and therapeutic decisions. In recent years, insights into the complex network of molecular markers underlying this diversity have shown marked progress due to the detection of novel mutations, such as nucleophosmin gene (NPM1) in AML, and due to the description of cooperation pathways in leukemogenesis. Also, targeted therapeutic strategies are continuously expanding as illustrated by the tyrosine kinase inhibitor (TKI) imatinib for BCR-ABL positive ALL. Thus, molecular analysis based on various techniques, such as polymerase chain reaction (PCR) has become an essential part of the diagnostic panel for acute leukemia. In addition, cytomorphology, cytogenetics, fluorescence in situ hybridization (FISH), and immunophenotyping with multiparameter flow cytometry (MFC) need to be applied for diagnosis. During the course of disease, the residual leukemic cell load can be monitored by highly sensitive quantitative PCR techniques ("real-time PCR"). At present, new techniques, such as high throughput sequencing (next generation sequencing, NGS) or gene expression profiling with microarrays are being explored for use in hematological malignancies, and are being evaluated in preclinical studies. This demonstrates that molecular diagnostics for acute leukemias are in continuous development. This review summarizes the most important recurrent molecular markers seen in acute leukemias, their role in prognosis and therapy and provides an overview on the relevant PCR techniques.