The Paramecium aurelia complex, a group of morphologically similar but sexually incompatible sibling species, is a unique example 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 have 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 5 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 non-functional mtA alleles, and identify novel developmental rearrangements in mtA and mtB which now explain all cases of maternally inherited mating-type determination. Epistasis between these genes likely evolved from less specific interactions between paralogs in the P. aurelia common ancestor, after a whole-genome duplication, but the mtB gene was subsequently lost in three P. aurelia species which appear to have returned to an ancestral regulation mechanism. These results suggest a model accounting 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.