The immunoglobulin variable heavy chain (IgV_H) gene mutation status is an important prognostic factor in chronic lymphocytic leukemia (CLL), since cases with mutated V_H genes show significantly longer survival than unmutated cases. Recently, we reported a preferential use of the V_H3-21 gene in mutated CLL and showed that mutated V_H3-21 cases had an inferior overall survival compared with other mutated CLL. In order to further characterize this subset, we performed V_H gene analysis in 265 CLL cases and identified 31 V_H3-21 cases (11.7%); 21 cases had mutated and 10 cases unmutated V_H genes. Regardless of V_H gene mutation status, a poor overall survival was found in the V_H3-21 cases with a median survival of 83 months. These survival data confirm that V_H3-21 cases do not fit into the general prognostic grouping of mutated and unmutated CLL. A large fraction of V_H3-21 cases also demonstrated unique features with shorter lengths of the third complementarity determining region (CDR3) and CDR3s with highly homologous amino acid sequences. Furthermore, the V_H3-21 cases showed a striking dominance of λ light chain expression, and analysis of the Igλ gene rearrangements revealed highly restricted use of the V_λ2-14/J_λ3 genes in the majority of cases. Taken together, our new findings strengthen the suggestion that V_H3-21–using cases comprise a new CLL entity, irrespective of V_H gene mutation status, and implicate that a common antigen epitope, perhaps of pathogenic significance, is recognized by the highly homologous V_H3-21/V_λ2-14 Ig molecules expressed in individual tumors.