De novo CD5-positive (CD5+) diffuse large B-cell lymphoma (DLBL) has recently been identified as constituting a homogeneous subgroup with distinct clinicopathologic and genotypic characteristics, but its origin remains to be elucidated. Previous studies by sequence analysis of the variable region of the immunoglobulin heavy chain (VH) have shown that CD5+ B-cell malignancies such as mantle cell lymphoma (MCL) and B-cell chronic lymphocytic leukemia (B-CLL) cells represent pre-germinal center (pre-GC) stage B cells in contrast with the post-GC stage of most DLBLs, which show somatic hypermutations in VH genes. In the present study, we investigated the VH sequence of de novo CD5+ DLBL to clarify whether CD5+ DLBL represents the pre-GC stage, as do other CD5+ B-cell malignancies, or the post-GC stage, as is typical of DLBL. All eight cases (four CD5+ DLBL and four CD5-negative (CD5-) DLBL) examined by us showed somatic hypermutations in the VH segment and two of the CD5- DLBL cases showed intra-clonal diversity, suggesting that CD5+ DLBLs were derived from the same maturation stage as CD5- DLBL, but were distinct from the other indolent CD5+ B-cell lymphomas of B-CLL and MCL. These data suggest that de novo CD5+ DLBLs do not merely lie within a continuous spectrum with B-CLL and MCL, but represent a biologically distinct variant within the diagnostic framework of diffuse large B-cell lymphoma.