Hairy cell leukemia being defined as a mature B-cell lymphoma shows unique clinicopathological, immunophenotype, and genetic alteration features among another mature B-cell malignancy. Although lymphocytes with hairy appearance and co-expression of CD25, CD11c and CD103 markers on B-cells are compatible with hairy cell leukemia, there are some difficulties in differential diagnosis between HCL with its variant and splenic marginal zone lymphoma. The presence of kinase-activating BRAF V600E mutation in classical HCL clone and in no other mature B-cell lymphomas/ leukemia has implication in differential diagnosis. In this study, we evaluated BRAF V600E mutation detection of H&E stained peripheral blood smears by Sanger sequencing method in patients with classical HCL and findings related to morphology and immunophenotype. Peripheral smear review indicated a subpopulation of atypical lymphocytes with villous cytoplasmic projections. By flow cytometry analysis, B-cells represented immunophenotype of CD19+ CD20(bright)+ CD11c+ CD25+ CD103+. Sanger sequencing demonstrated that presence of BRAF V600E mutation in 86.6% (13/15) of analyzed HCL DNA. Hence, gain-of-function mutation of BRAF V600E is detectable in DNA of peripheral blood smear of patients with classical HCL that were evaluated by immunophenotyping. In conclusion, DNA from peripheral blood smear can be used to identify BRAF V600E mutation using Sanger sequencing method in samples with at least 10% HCL clone as supplementary approach to aid in the diagnosis of classical HCL.