Silene latifolia and S. dioica are model systems in studies of plant reproduction, chromosome evolution and sexual dimorphism, but sexing of plants based on morphology is only possible from flowering stage onwards. Both species show homogametic females (XX) and heterogametic males (XY).•Here we developed two assays (primer pairs ss816 and ss441) for molecular sexing of S. latifolia and S. dioica, targeting length polymorphisms between the X and Y-linked copies of the spermidine synthase gene.The two assays were successful in identifying known (flowering-stage) males and females from UK and Spanish populations, with an error rate of 3.1% (ss816; successful for both species) and 0% (ss441, only successful for S. latifolia). Our assays therefore represent novel tools for rapid, robust and simple determination of the genotypic sex of S. latifolia and S. dioica.