In primary myelofibrosis (PMF) and essential thrombocythemia (ET) the megakaryocytic lineage characteristically shows aberrant proliferation and maturation in which the regulatory microRNA (miR) system might be involved. Laser-microdissected PMF and ET megakaryocytes were analysed with real-time polymerase chain reaction (PCR) low density arrays comprising 365 microRNAs. The highest megakaryocytic expression levels were observed for miR-223, which is known to be expressed also in granulopoiesis. Cluster analysis revealed a tendency of disease-specific megakaryocytic microRNA expression profiles indicating that a complex shift of microRNA expression appears to be the underlying defect. Increased accumulation of miR-146b was observed in cellular stage PMF (p = 0.0125) but not ET megakaryopoiesis. In conclusion, this is the first microRNA profiling of in situ-derived PMF, ET and normal megakaryocytes.