The classical Philadelphia chromosome-negative myeloproliferative neoplasms consist of three main pathological and clinical entities with the recurrent JAK2 V617F mutation present in ∼98% of patients with polycythemia vera and ∼50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The recent discovery of mutations within the CALR gene in up to 80% of JAK2 V617F-negative ET and PMF patients compels employment of CALR mutational analysis for the molecular diagnosis of these diseases. Evidence derived from a retrospective audit of JAK2 V617F testing provides a framework for the proposal of a diagnostic algorithm that incorporates CALR mutation assessment. It is recommended that relevant clinical details, including a provisional morphological and clinical diagnosis, are provided and that exclusion of alternative causes of erythrocytosis, leucocytosis, or thrombocytosis is required before molecular testing. It should be acknowledged that over-requesting such investigations can impact on the clinical predictive value of these tests when considering the disease specificity of such mutations, with adherence to the diagnostic algorithm necessary to ensure appropriate use of resources.