The anther smut fungus Microbotryum violaceum ( = Ustilago violacea) is a parasite of many species of the Caryophyllaceae. Its host specificity has been debated since early this century, when cross-inoculation experiments indicated the existence of host-specific lineages. Recently, on the basis of spore ultrastructure, all presumed host races were lumped within M. violaceum. To measure gene flow among natural populations of anther smuts from different host species, we used microsatellite variation at 5 loci among samples from 8 Silene, 2 Saponaria, 2 Dianthus and 1 Gypsophila species. Most of the 326 M. violaceum samples investigated originated from the Swiss Alps and close surroundings. Microsatellite variation revealed almost perfect isolation among anther smut fungi from different host species. In addition, differentiation was supported by the nonrandom distribution of null alleles among samples from different host species and host genera. Null alleles were most abundant in anther smut samples from non-Silene hosts. The resolution of genetic differentiation among anther smuts from different host species was highest in those from Silene species. Genetic relationships among samples as indicated by Neighbour-Joining analysis based on genetic distances are discussed with respect to host phylogeny and host ecology. One sample was identified as Ustilago gausseni because it had verrucose instead of reticulate spores and was collected from Silene italica. Neighbour-Joining analysis revealed that this sample was similar to the M. violaceum samples from other Silene host species. Therefore, our data question spore morphology as a reliable character for anther smut systematics.