FMS-like tyrosine kinase 3 receptor- internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia (AML) and is considered rare in acute lymphocytic leukemia (ALL). Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic PCR and have recently incorporated GeneScan to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed AML (n=5) and ALL (n=3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. 7/8 patients considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis (SCA). Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested, provides an evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity (LOH) of the normal allele, affecting 25-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.