Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised patients. To investigate the genetic diversity of P. carinii populations, multilocus enzyme electrophoresis was used to analyze five enzyme systems (malate dehydrogenase, glucose phosphate isomerase, leucine aminopeptidase, malic enzyme, and 6-phosphogluconate dehydrogenase). Only five different multilocus associations (zymodemes) were recorded for the 70 isolates studied. While only one multilocus combination was found in mice and rabbits, three different multilocus associations were recorded in rats. Population genetic tests and phylogenetic analysis strongly suggest that P. carinii genotypes are host-specific, in agreement with molecular study results, and that no genetic exchange occurs between genotypes from different host species. This hypothesis could be verified only by the evolutionary genetic approach, which relies here on multilocus analysis.