Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs in all 91 stocks of twelve New and Old World Leishmania species examined; related sequences are present in some other kinetoplastid species. LD1 has no homology to several DNA sequences that are amplified in drug-resistant Leishmania. LD1 occurs in 3 different genomic organizations in Leishmania, depending on the stock. It is present within large (1.5-2 megabase) chromosomes in all stocks, and 74 stocks contain only this form. In 12 other stocks, LD1 also occurs in smaller (less than 550 kb) chromosomes, some of which are multicopy. Five stocks contain LD1 in multicopy circular DNA molecules in addition to the sequences found in the larger chromosome(s). Restriction fragment length polymorphisms of LD1 sequences correlate with taxonomic grouping, suggsting that LD1 is an endogenous sequence.