The detection of incipient host-shifts is important to the study of emergent diseases because it allows the examination of ecological and genetic conditions that favor novel inter-species transmission. Mixed populations of Silene latifolia and Silene vulgaris were investigated for the putative occurrence of host-shifts by the fungal plant pathogen Microbotryum violaceum (the cause of anther-smut disease) between S. latifolia (a common host for the pathogen) and S. vulgaris (a rare host). Samples of the fungus from mixed and pure host populations were studied for variation in their electrophoretic karyotypes. A karyotype distance matrix showed that fungal samples clustered by locality, but not by host species. Fungal samples from S. vulgaris were indistinguishable from sympatric samples from S. latifolia in multiple cases. The results indicated at least two independent host-shifts, one in the US and perhaps two in Italy. The karyotype and ecological data indicate that the direction of the host-shifts is from S. latifolia to S. vulgaris.