The investigation of microsatellite markers has recently superseded that of isoenzymes for many population-biology applications. Microsatellites have the advantages of being dominant, neutral, highly polymorphic and easily scored by high-throughput methods. However, it is necessary to develop a new panel of markers for each group of organisms of interest. Previously, only about 5% of the markers that amplify Leishmania major microsatellite loci were also found to amplify L. donovani loci. A panel of 20 microsatellite markers that are polymorphic in L. donovani and L. infantum has now been developed, using a rapid-enrichment method that will be suitable for developing libraries of markers for other trypanosomatid species. This is the first panel of polymorphic microsatellite markers, to be isolated de novo from any species of Leishmania, that is large enough for population-biology applications.