We have used a chromosome-specific approach to generate a 300 kb long ‘contig’ across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The ‘contig’ generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb ‘contig’ rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.