We evaluated CD123 expression in 95 pediatric and 24 adult ALL patients and compared the results with the CD123 expression in normal B-cell precursors. Early B-cell precursors were negative while intermediate precursors and mature B cells showed weak CD123 expression. Leukemic blasts in 31% of precursor-B ALL samples exhibited strong expression of CD123, 61% had moderate CD123 expression and 8% were negative; 81.5% of ALL with hyperdiploid karyotype (≥ 52 chromosomes) showed strong CD123 overexpression. In contrast, cases with ETV6/RUNX1 rearrangement had weak CD123 expression. Our study suggests that overexpression of CD123 is an aberrant phenotype present in a subset of precursor-B ALL with hyperdiploid genotype, and represents an additional marker of good prognosis in pediatric precursor-B ALL. Moreover, aberrant CD123 expression in ALL is a good marker for monitoring of minimal residual disease.