In acute lymphoblastic leukemia (ALL) patients, automated fluorescence fragment analysis (ALF) has been reported to improve the monoclonality detection rate of immunoglobulin heavy chain genes (IgH) polymerase chain reaction (PCR) analysis. This study performed complementary determining region (CDR) I and III PCR on samples from 135 patients with B-cell neoplasias and 25 healthy controls. The value of ALF was investigated in comparison to the widely used ethidium bromide (ETB)-stained agarose gels (AGGE). ETB-stained AGGE detected monoclonal CDR III PCR products in 53/72 ALL, in 22/34 non-Hodgkin's lymphoma (NHL), 13/22 multiple myeloma (MM), and 2/7 monoclonal gammopathies (MGUS). ALF identified monoclonal CDR III amplificates in 55/72 ALL, 23/34 B-NHL, 14/22 MM, and 2/7 MGUS. AGGE achieved clonal CDR I PCR results in 30/64 samples, while ALF detected 34 clonal CDR I product patterns. Taking together, ETB-stained AGGE revealed monoclonality in 120/199 PCR products versus 129/199 by ALF. Compared with AGGE and ETB-staining, ALF offers a slightly increased sensitivity and can be recommended for the evaluation of difficult samples.