When the female plant of Silene latifolia is infected with the smut fungus Microbotryum violaceum, its rudimentary stamens develop into anthers which contain fungus teliospores instead of pollen. To identify genes required for maturation of anthers in S. latifolia, we performed a cDNA subtraction approach with healthy male buds and female buds infected with M. violaceum. We isolated five cDNA clones, which were preferentially expressed in healthy male buds during stages associated with a burst in tapetal activity. These five cDNAs are predicted to encode a mandelonitrile lyase protein (SlMDL1), a strictosidine synthase protein (SlSs), a glycosyl hydrolase 17 protein (SlGh17), a proline-rich protein APG precursor (SlAPG), and a chalcone-synthase-like protein (SlChs). All five genes showed expression in both healthy and fungus-infected male buds, but not expressed in either healthy or infected female buds. The first three genes were highly expressed in both tapetum and pollen grains while the last two genes were expressed only inside the tapetum of male flower buds. Phylogenetic analysis results showed that SlChs and SlGh17 belong to anther-specific subgroups of chalcone-synthase-like genes and glycosyl hydrolase 17 family genes, respectively. Our results suggest that the isolated five genes are related to the fertility of the anther leading to the development of fertile pollen. The smut fungus was not able to induce the expression of the five genes in the infected female buds. This raises the possibility that these genes are under the control of master gene(s) on the Y chromosome.