The Paramecium aurelia complex, a group of morphologically similar but sexually incompatiblesibling species, isa uniqueexample of the evolutionary plasticity of mating-type systems. Each species has two mating types, O (Odd) and E (Even). Although O and E types are homologous in all species, three different modes of determination and inheritance have been described: genetic determination by Mendelian alleles, stochastic developmental determination, and maternally inherited developmental determination. Previous work in three species of the latter kind has revealed the key roles of the E-specific transmembrane protein mtA and its highly specific transcription factor mtB: type O clones are produced by maternally inherited genome rearrangements that inactivate either mtA or mtB during development. Here we show, through transcriptome analyses in five additional species representing the three determination systems, that mtA expression specifies type E in all cases. We further show that the Mendelian system depends on functional and nonfunctional mtA alleles, and identify novel developmental rearrangements in mtA and mtB which now explain all cases of maternallyinheritedmating-typedetermination.Epistasisbetweenthesegeneslikelyevolvedfromlessspecificinteractionsbetween paralogs in the P. aurelia common ancestor, after a whole-genome duplication, but the mtB gene was subsequently lost in three P. aurelia specieswhichappear tohave returnedtoanancestralregulationmechanism. Theseresultssuggesta modelaccounting for evolutionary transitions between determination systems, and highlight the diversity of molecular solutions explored among sibling species to maintain an essential mating-type polymorphism in cell populations.