Abstract: Recent studies have demonstrated that B‐cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M‐CLL) or unmutated (UM‐CLL) immunoglobulin variable heavy‐chain (V_H) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the V_H gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the V_H genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM‐CLL and M‐CLL patients. The similarity of M‐CLL and UM‐CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogenous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M‐CLL and UM‐CLL. More positive cells in the UM‐CLL group were observed regarding CD38, but CD38 was not a good predictor of V_H gene mutation status. Seventy percent of the M‐CLL cases, but only 36% of UM‐CLL cases, were Ig‐λ⁺. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M‐CLL, whereas the GMF intensity of CD45RA tended to be associated with UM‐CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.