| Summary: | <i>Phaffia</i> is an orange-colored basidiomycetous yeast genus of the order Cystofilobasidiales that contains a single species, <i>P. rhodozyma</i>. This species is the only fungus known to produce the economically relevant carotenoid astaxanthin. Although <i>Phaffia</i> was originally found in the Northern hemisphere, its diversity in the southern part of the globe has been shown to be much greater. Here we analyze the genomes of two Australasian lineages that are markedly distinct from <i>P. rhodozyma</i>. The two divergent lineages were investigated within a comprehensive phylogenomic study of representatives of the Cystofilobasidiales that supported the recognition of two novel <i>Phaffia</i> species, for which we propose the names of <i>P. australis</i> sp. nov. and <i>P. tasmanica</i> sp. nov. Comparative genomics and other analyses confirmed that the two new species have the typical <i>Phaffia</i> hallmark—the six genes necessary for the biosynthesis of astaxanthin could be retrieved from the draft genome sequences, and this carotenoid was detected in culture extracts. In addition, the organization of the mating-type (<i>MAT</i>) loci is similar to that of <i>P. rhodozyma</i>, with synteny throughout most regions. Moreover, cases of trans-specific polymorphism involving pheromone receptor genes and pheromone precursor proteins in the three <i>Phaffia</i> species, together with their shared homothallism, provide additional support for their classification in a single genus.
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