Most dioecious plant species are believed to derive from hermaphrodite ancestors. The regulatory pathways that have been modified during evolution of the hermaphrodite ancestors and led to the emergence of dioecious species still remain unknown. Silene latifolia is a dioecious plant species harboring XY sex chromosomes. To identify the molecular mechanisms involved in female organ suppression in male flowers of S. latifolia, we looked for genes potentially involved in the establishment of floral organ and whorl boundaries. We identified homologs of Arabidopsis thaliana SHOOTMERISTEMLESS (STM) and CUP SHAPED COTYLEDON (CUC) 1 and CUC2 genes in S. latifolia. Our phylogenetic analyses suggest that we identified true orthologs for both types of genes. Detailed expression analyses showed a conserved expression pattern for these genes between S. latifolia and A. thaliana, suggesting a conserved function of the corresponding proteins. Comparative in situ hybridization experiments between male, female, and hermaphrodite individuals reveal that these genes show a male-specific pattern of expression before any morphological difference become apparent. Our results make SlSTM and SlCUC strong candidates for being involved in sex determination in S. latifolia.