New collections are reported of the monospecific genus Polypyrenula, an apparently extinct and doubtfully lichenized fungus, typically classified in the Pyrenulaceae. Anatomical studies reveal that it is facultatively lichenized. The structure of its hamathecium suggests affinities with Dothideomycetes rather than Eurotiomycetes. Molecular analysis using nuLSU and mtSSU markers demonstrates for the first time its inclusion in Trypetheliaceae, outside the core genera as part of the early diverging lineages in this family. The known distribution of Polypyrenula is extended to Mexico and South America, new information on its phorophyte associations is provided, and the name Polypyrenula sexlocularis is reinstated as the correct name for this species.
