Nectar inhabiting yeasts are commonly found in many plant species. Even though these microorganisms are abundant, much is still unknown about the communities of these microorganisms within and among plant species as well as the diversity of these microorganisms within and among seasons. This study examines the presence and diversity of these microorganisms in the dioecious plant species Silene latifolia as well as coflowering plants in Virginia, U.S. The majority of previous research on nectarivorous yeasts has been on hermaphroditic plant species. This is one of the few studies examining a dioecious plant species. Yeast species were isolated from the floral nectar of S. latifolia and associated plant species in 1999–2001, morphotyped into 37 morphospecies, and identified into 26 operational taxonomic units (OTUs) by sequencing the D1/D2 domain of the large subunit nrDNA. Yeast species from ascomycetes and basidiomycetes were isolated from nectar, including nectar associated ascomycetous Metschnikowia species and the basidiomycetous anther smut Microbotryum violaceum; however, ascomycetes dominated the samples. Metschnikowia species only accounted for 21% of the yeast isolates, a much lower percentage than in other studies. There was evidence of both host specificity and widespread dispersal of yeast species with Microbotryum violaceum found in nectar from both healthy and consistently diseased plant populations, indicating long distance dispersal of this fungus. There were no consistent differences among the sexes of S. latifolia, but male floral nectar appeared to have a higher diversity and species richness in some years. There was some indication of seasonality of yeast abundance in nectar as well as differences among years, potentially due to drought conditions. These results support the current body of evidence by researchers that, although not diverse in terms of the number of different yeast species found in nectar, yeasts are abundant in the nectar of plants.