An acquired mutation in Janus kinase 2 (JAK2), V617F, has recently been identified in human myeloproliferative disorders. Detection of the mutation is helpful in differential diagnosis, prognosis, and predication of therapeutic response. Because the mutation can be present in a small proportion of granulocytic populations in myeloproliferative disorder patients, a highly sensitive detection method is required. In this study, we systematically optimized the reaction conditions of a published amplification refractory mutation system-polymerase chain reaction research protocol to make it a robust clinical diagnostic test. The modifications led to a clear demonstration of the V617F mutation in a patient who would have been easily missed by the original amplification refractory mutation system-polymerase chain reaction assay. The test detects the V617F mutation not only with a high analytic sensitivity of 0.05 to 0.1% but also with a high diagnostic specificity of 99%. In addition, the assay has the ability to distinguish cases with only mutant alleles from cases with mixed normal and mutant alleles. The assay is fast and easy to perform, and no special equipment other than thermocyclers is required. All these features make the assay readily and broadly applicable in clinical molecular diagnostic laboratories.