Acute lymphoblastic leukemia (ALL), the most common cancer in childhood, is characterized by clonal proliferation of transformed lymphoblasts that comprise the majority of marrow and/or blood specimens. Although the leukemic cells typically express antigens associated with lymphoid maturation or activation (ie CD19, CD38, etc), it has been suggested that ALL blasts may evolve from a more primitive precursor. Increased understanding of the phenotypic and molecular heterogeneity of cells in ALL may provide clues to leukemogenesis and/ or impact prognostication or treatment. We utilized a phenotype/genotype approach to measure the prevalence and frequency of cytogenetically aberrant cells in a phenotypically defined primitive compartment (CD34+33-19-38-; CD34+Lin-). Bone marrow cells were flow cytometrically sorted into CD34-Lin+, CD34+Lin+ and CD34+Lin- subpopulations. Fluorescence in situ hybridization (FISH) was used to quantify the frequency of cells with aneusomies in the sorted populations. Approximately 26% (5/19) of ALL cases at diagnosis contain cytogenetically aberrant CD34+Lin- cells. The frequency of cytogenetically aberrant cells in the CD34+Lin- compartment is independent of FAB, WBC and blast counts. These data indicate that cytogenetically aberrant cells may reside in a phenotypically defined primitive subpopulation and suggest that ALL blasts in some patients may evolve from a precursor compartment.