Abstract: We describe here the rationale, implementation and results of a bioinformatic strategy for large-scale identification and annotation of chromosomal translocations in tumours, based on sequence and annotation comparison between human transcriptome and EST partial cDNA sequences derived from tissues or cell lines. We also illustrate how the sequencing and subsequent careful bioinformatic analysis of a number of identified candidate translocation cDNAs revealed the complexity of distinguishing recombination from true translocation events. Finally, we suggest some EST filtering and cleaning strategy for pursuing EST-based “in silico” translocation identification projects.