The somatically acquired V600E mutation of the BRAF gene has been recently described as a molecular marker of hairy cell leukemia (HCL). We developed an allele-specific PCR for this mutation and studied 62 patients with HCL, 1 with HCL variant, 91 with splenic marginal zone lymphoma, 29 with Waldenström macroglobulinemia, and 57 with B-cell chronic lymphoproliferative disorders. The BRAF V600E mutation was detected in all HCL cases and in only 2 of the remaining 178 patients. These 2 subjects had B-cell chronic lymphoproliferative disorders that did not fulfill the diagnostic criteria for HCL. Despite the positive PCR finding, the mutation could not be detected by Sanger sequencing in these 2 cases, suggesting that it was associated with a small subclone. We conclude that the BRAF V600E mutation is present in all patients with HCL and that, in combination with clinical and morphologic features, represents a reliable molecular marker for this condition.