In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology. © 2006 Cambridge University Press.