ABSTRACTPneumocystis cariniiis an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity ofP. carinii.These additions provide new understanding ofPneumocystistransmission and the effect of possible reservoirs ofPneumocystisin the various species. In this study, a 400‐bp fragment of the thymidylate synthase (TS) gene ofP. cariniihas been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat‐derivedP. cariniiwas hybridized with the amplified products from rat‐ and mouse‐derivedP. carinii, but not with rabbit or humanP. cariniiDNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and humanP. cariniiwere determined. Differences were detected in the gene fragment inP. cariniiisolates from the 4 host species; however no difference was revealed inP. cariniiisolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of theP. cariniiTS gene appeared as host‐species specific. A specific probe which recognized all humanP. cariniiisolates wa