The nearly one hundred species of Euplotes evolved high-multiple mating systems. Based on analyses of Mendelian genetics, these systems have for long been assumed to be uniformly determined by series of multiple alleles inherited at a single micronuclear mat locus. The chemical characterization in an array of Euplotes species of water-borne signaling proteins (pheromones) specified by the macronuclear (transcriptionally active) versions of the micronuclear (transcriptionally inert) mat alleles has permitted us to evolve in the study of the biology of Euplotes mating types from an approach of Mendelian genetics to an approach of molecular genetics. We have cloned and structurally characterized families of macronuclear pheromone (mat) genes from a number of species that take different positions in the Euplotes phylogenetic tree. In accord with the prediction from Mendelian genetics, it appeared that early branching species, such as E. polaris, E. raikovi and E. nobilii, inherit their mating types at a single multi-allelic mat locus. However, in disagreement with the prediction from Mendelian genetics, late branching species, such as E. crassus and E. focardii, inherit their mating types at two distinct mat loci that are likely the result of an event of gene duplication in the micronuclear genome. One locus appears to be functionally homologous with the multi-allelic mat locus of the early branching species and is thus deputed to synthesize cell type-specific pheromones. The second locus, instead, appears to be functionally divergent and deputed to synthesize pheromones that are structurally identical among restricted sets of mutually mating compatible cell types.