We have used a chromosome fragmentation strategy based on systematic genomic insertions of the rare cutting yeast I-SceI endonuclease to assess structure and sequence organization of homologous chromosomes between evolutionary divergent pathogenic and non-pathogenic Leishmania species. This method was combined to physical mapping and hybridization studies using a number of specific chromosomal markers as probes. Our studies have concentrated on two different chromosomes of Leishmania major (L. major), L. donovani and L. infantum and of the non-pathogenic species L. tarentolae. Specific chromosome fragmentation events at the level of multiple I-SceI genomic integrations indicated that very similar distances separated internal genomic sequences between homologous chromosomes and that distances from chromosome ends were more variable. The order and orientation of genes along the homologous chromosomes were also conserved between species. With only few exceptions, genome organization between pathogenic and non-pathogenic Leishmania species was found to be highly conserved. Genomic comparison of pathogenic and non-pathogenic species may be useful for depicting regions involved in species-specific related pathologies. (C) 2000 Elsevier Science B.V. All rights reserved.