Analysis of clonotypic immunoglobulin M (IgM) from 15 patients with Waldenstrom macroglobulinemia (WM) showed a strong preferential use of the V_H3/J_H4 gene families. Identification of the WM IgM V/D/J was validated using single-cell analysis, confirming its presence in most B cells. Despite the extensive hypermutated V_H genes in 13 of 15 patients, statistical analysis of framework/complementary-determining region (FR/CDR) mutation patterns suggests that they might have escaped antigenic selection. Neither intraclonal diversity nor isotype switching was detectable. Membranous and secreted forms of clonotypic IgM transcripts were present in bone marrow and blood. Single-cell analysis showed that clonotypic B cells coexpress CD20, surface IgM (sIgM), and sIgD but that they lack CD138. Most B cells lacked memory marker CD27 despite their hypermutated variable regions otherwise suggestive of memory status. At diagnosis, circulating B cells in WM are largely clonotypic. However, when monoclonal IgM levels are decreased, clonotypic frequencies are substantially reduced despite elevated CD20⁺ cells, shown to be polyclonal by DNA sequencing and CDR3 fragment analysis. Thus, WM includes the expansion of circulating, polyclonal B cells. Overall, this work suggests that WM may originate from a largely V_H3-restricted, somatically mutated, predominantly CD27^-IgM⁺IgD⁺ population that cannot undergo class switching, suggestive of B cells that might have bypassed the germinal center. (Blood. 2004;104:2134-2142)