Internal tandem duplication (ITD) mutations of the FLT3 gene have been associated with inferior prognosis of acute myeloid leukemia. Detection of minor clones or minimal residual clones with ITD mutations is desirable, but is challenging when the mutant signal determined by polymerase chain reaction (PCR) and capillary electrophoresis is weak. In this study, we applied delta-PCR, which is a triple-primer strategy, to ensure PCR specificity and improve the sensitivity to 0.1% leukemic cells with ITD mutation. We also applied a reference peak to calculate ITD allelic burdens of <2% threshold of technical limitation for evaluating the relative ratio of 2 signals by capillary electrophoresis. Delta-PCR was able to detect single or multiple ITD mutations with an allelic burden (peak height ratio of mutant allele and wild-type allele) ranging from 0.4% to >100% among all 31 cases with previous documented ITD mutations. In one of the 3 cases with previously reported negative ITD mutation in the initial diagnostic specimen and ITD-positive results in the follow-up specimens, an ITD of 0.04% allele burden was retrospectively detected in the initial diagnosis specimen using delta-PCR. We also demonstrated that minor ITD mutant clones with an allelic burden of <1% present at diagnosis may become a dominant clone at the later refractory status, suggesting that detection of leukemic clones with allelic burdens of <1% may be clinically significant. Delta-PCR can detect ITD mutations with improved sensitivity and specificity and may be useful for the detection of minimal residual leukemia.