Ciliates are a diverse group of microbial eukaryotes that exhibit tremendous variety in several aspects of their mating systems. To understand the evolutionary forces driving mating system diversification in ciliates, we use a comparative approach synthesizing data from many ciliate species in light of recent phylogenetic analyses. Specifically, we investigate the evolution of number of mating types, mode of mating type inheritance, and the molecular determinants of mating types across the taxonomic diversity of ciliates, with an emphasis on three well-studied genera: Tetrahymena, Paramecium, and Euplotes. We find that there have been many transitions in the number of mating types, and that the requirement of nuclear reorganization may be a more important factor than genetic exchange in determining the optimum number of mating types in a species. We also find that the molecular determinants of mating types and mode of inheritance are evolving under different constraints in different lineages of ciliates. Our results emphasize the need for further detailed examination of mating systems in understudied ciliate lineages. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 187–197.