The screening for JAK2 V617F mutation in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis offers crucial information for the final diagnosis of these disorders. Recently, several JAK2 exons 12 and 14 mutations have been detected in V617F-negative patients with idiopathic erythrocytosis. The need for a rapid and accurate assay for the mutation screening in both exons 12 and 14 prompted us for the application of a method for the analysis of the entire coding region between exons 11 and 15. We applied the non-isotopic RNase cleavage assay and the accuracy of the method was verified in a series of V617F-positive, V617F-negative patients and healthy individuals, with no false results. This method can be applied in any laboratory without the requirement of specific sophisticated equipment.