Partial nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences (953 and 385 nucleotides, respectively) of one fish monogenean (outgroup) and six polystome monogeneans (four Polystomoides spp. from the oral cavities and urinary bladders of freshwater turtles in Australia and Malaya, two Neopolystoma spp. from the urinary bladder and conjunctival sac of a freshwater turtle in Australia) were used to examine the question of whether congeneric species infecting different sites in the same host species have speciated in that host by adapting to different sites, or whether species infecting a particular site in one host have given rise to species infecting the same site in different hosts. Results show unequivocally that congeneric species infecting the same site, even of host species belonging to different suborders and occurring on different continents, are more closely related than congeneric species infecting different sites of the same host species. This is interpreted as meaning that speciation has not occurred in one host. Morphological evolution of polystomes has been very slow: few differences between species and even genera have evolved over a period of at least 150 Myr, and this is matched by low substitution rates of nucleotides, and the ambiguous position of species of different genera, depending on whether COI or 28S rDNA sequences are used.