The evaluation of molecular and cytogenetic characteristics using novel techniques has significantly contributed into the insight of leukemia. In this study, immunoglobulin heavy chain gene rearrangements (V(H)D(H)J(H) region) were analyzed by polymerase chain reaction (PCR). Point mutations of the D835/I836 in the activation loop (AL) domain of the second tyrosine kinase domain of the fms-related tyrosine kinase 3 (FLT3) gene and NRAS (neuroblastoma cell line) point mutations were also analyzed by PCR. Furthermore, sequence analysis of the V(H)D(H)J(H) region was performed, as well as, chromosomal aberrations were identified by interphase fluorescence in situ hybridization (iFISH) in a 12.5-year-old boy with acute lymphoblastic leukemia. Positive MRD was found in bone marrow samples obtained at various time points during and after treatment completion prior to relapse. Molecular analysis of the FLT3 gene mutations revealed an acquired a G → T mutation at codon 835, which resulted to substitution of aspartate 835 for tyrosine (D835Y). Cytogenetic analysis with iFISH showed t(9;22) (q34;q11.2), with minor-BCR/ABL1 fusion gene in the majority of nuclei, while a subclone with duplication of the Philadelphia chromosome was observed. Triple signals of AML1 were detected in 80% of nuclei, which were compatible with trisomy of chromosome 21. BCR/ABL1 fusion gene, duplication of Philadelphia chromosome and persistence of MRD constitute inferior prognostic factors, while hyperdiploidy, trisomy of chromosome 21 and FLT3-AL mutations are related to better prognosis. The study of cytogenetic and molecular characteristics is essential in order to decide on the optimal treatment protocol in childhood leukemia.