The diagnosis of the BCR-ABL-negative myeloproliferative neoplasms (MPN), namely polycythemia vera, essential thombocythemia and primary myelofibrosis has relied significantly on the detection of known causative mutations in the JAK2 or MPL genes, which account for the majority of MPN patients. However, around 30 % of patients with MPN, primarily essential thombocythemia and primary myelofibrosis, lack mutations in these two genes making it difficult to reach a confident diagnosis in these cases. The recent discovery of frameshift mutations in CALR in approximately 70 % of MPN patients lacking the JAK2 and MPL mutations offers a reliable diagnostic marker for the latter group. A review of the current literature, plus unpublished data from our laboratory, shows that 55 different CALR insertion/deletion mutations have been identified so far in MPN patients. Among these 55 variants reported to date, a 52-base pair deletion and a 5-base pair insertion are by far the most prominent representing 50 and 35 %, respectively, of all cases with CALR mutations. In this paper, we describe a high-resolution melting (HRM) analysis and a Taqman® Real-Time PCR (RQ-PCR) assay and we propose a new clinical laboratory diagnostic algorithm for CALR mutation analysis. According to this algorithm, samples can go through front-line screening with HMR or fragment analysis, followed by the newly developed RQ-PCR to both discriminate and quantify the two most common mutations in CALR gene.