Chromosomal rearrangements are recurrent findings in human cancer and result in aberrant restructuring of the genome. The majority of known fusion genes are the consequence of reciprocal (balanced) translocations. However, most translocations described in human cancer are unbalanced, suggesting that other cancer genes remain to be identified. Historically, it was assumed that these unbalanced rearrangements affected gene function through the loss or gain of chromosomal material. However, emerging data supports direct disruption of genes located at or close to the unbalanced translocation breakpoints. New approaches are required for the identification of those gene loci underlying unbalanced translocations in cancer, as traditional methods have had limited success. This review focuses on one such strategy, using traditional and innovative molecular technologies to characterize breakpoint heterogeneity within a series of acute lymphoblastic leukemia (ALL) patients with dicentric chromosomes. This approach has shown that in ALL, specific gene loci can be targeted by heterogeneous translocation breakpoints involving multiple partner chromosomes. Carcinomas have a high proportion of unbalanced rearrangements and relatively few significant genes have been identified. The application of the same strategy to their analysis will lead to the discovery of novel cancer genes and improve our understanding of the genetic basis of tumorigenesis.