Introduction Chronic lymphocytic leukemia is usually diagnosed through the characteristic morphology/immunophenotype of the lymphocytes, but some CLL cases remain atypical resulting in diagnostic uncertainty. Methods Using flow cytometry analysis, we investigated the expression of CDs160/200 on B cells from 124 patients (82 CLL, 42 other B-cell neoplasms) and nine controls. CDs160/200 measurements were determined as a ratio of the mean fluorescence intensities of leukemic cells/controls and were considered positive when the ratios were ≥2 and 20, respectively. Results Sixty and 83% CLL expressed CDs160/200 as compared to 5% and 10% of other B-cell neoplasms, respectively. None of the controls showed CDs160/200 expressions. Combination of both markers was observed in 55% of CLL but only in 2% of other B-cell neoplasms, and absence of both markers occurred in 12% of CLL but in 86% of other B-cell neoplasms. Conclusion CDs160/200 were associated with markers of the gold standard ‘Matutes score’ and could be useful markers to differentiate atypical CLL from other B-cell neoplasms in the absence of available biopsies or cytogenetics and molecular studies.