High hyperdiploidy (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) is characterized by non-random multiple trisomies and tetrasomies, involving in particular chromosomes X, 4, 6, 8, 10, 14, 17, 18, and 21. It is the most common cytogenetic subgroup in pediatric ALL, but in spite of this, the mechanisms behind its formation remain elusive. Four different pathways are possible: (1) initial near-haploidy followed by doubling of the chromosomes, (2) prior polyploidization with subsequent losses of chromosomes, (3) sequential gains of chromosomes in consecutive cell divisions, and (4) a simultaneous gain of all additional chromosomes in a single abnormal mitosis. Although these alternatives are difficult to distinguish experimentally, investigations of possible uniparental disomies (UPDs) and of the allelic ratios of loci located on tetrasomic chromosomes may provide some clues. In a previous study of 10 cases of hyperdiploid childhood ALL, we could exclude the first pathway as a general mechanism. Furthermore, the results did not favor the second alternative. Finally, our findings of equal allele dosage for chromosome 21 loci in cases with tetrasomy 21 suggested that the hyperdiploidy arose by a simultaneous gain of chromosomes. However, because all disomies and tetrasomies were not investigated, formation via a polyploid state or by a sequential gain could not be definitely excluded. In the present study, we have addressed this issue further by investigating 15 new cases of hyperdiploid childhood ALL using a total of 57 polymorphic microsatellite markers mapped to 23 of the 24 human chromosomes. Ten of the cases were analyzed with multicolor fluorescence in situ hybridization (M-FISH) and the remaining five with interphase FISH using probes for X, 4, 6, 8, 10, 14, 17, 18, and 21. Markers localized to all disomic and all tetrasomic chromosomes were then applied. One case displayed multiple UPDs and a non-typical pattern with only tetrasomies; this ALL probably arose via a near-haploid pathway. Two other cases had UPDs for 4/9 and 2/14 disomic chromosomes, respectively, and may have originated via a polyploid state. However, in the vast majority of the cases (12/15) there was no evidence for any UPDs, excluding that the hyperdiploidy originated by the near-haploid or polyploid mechanisms. Investigating the tetrasomies, 25 of a total of 27 tetrasomies present among the 15 cases displayed equal allele dosages, indicating a simultaneous gain of chromosomes as opposed to sequential gains. The two tetrasomies with unequal allele dosages were present in the same case, suggesting that this ALL constituted an exception. In conclusion, the results of the present study strongly suggest that hyperdiploidy in childhood ALL generally arises by a simultaneous gain of all additional chromosomes in a single abnormal mitosis.