Diverse JAK2 exon 12 mutations have been described in patients with V617F-negative polycythemia vera. Development of a sensitive detection assay capable of identifying any of these mutations is required for medium-throughput diagnostic screens. Non-mutated and mutant JAK2 exon 12 alleles were amplified from patient samples and cloned into plasmid vectors, then used to determine the sensitivity of a novel high-resolution melting-curve assay designed to detect all mutant JAK2 exon 12 alleles tested. High resolution melting analysis was more sensitive than direct sequencing and capable of detecting exon 12 mutations in granulocytes at moderate levels. In a blinded analysis of DNAs from V617F-negative erythrocytosis patients, with direct sequencing and allele-specific PCR used in one laboratory and high resolution melting analysis in another, high resolution melting successfully identified JAK2 exon 12 mutations in all 4 mutation-positive patients. High resolution melting analysis is a rapid, sensitive and high-throughput technique that is suitable for screening for JAK2 exon 12 mutations.