Two new genera are established in the Dothideomycetes based on morphological and molecular data (SSU and LSU nuclear ribosomal sequences) to accommodate four ascomycete species collected from woody debris submerged in freshwater habitats. The genus Minutisphaera is represented by a single species, M. fimbriatispora, which was collected from freshwater habitats in temperate forests in North America. It has small, superficial, brown, subglobose, papillate pseudothecia with dark, irregularly twisted hairs around the papillae, fissitunicate asci, septate pseudoparaphyses, and hyaline, one-septate ascospores surrounded by a gelatinous sheath and having spine-like appendages radiating around the ascospore at the septum. Minutisphaera formed a strongly supported clade with Farlowiella carmichaeliana. The second genus, Natipusilla, contains three new species, N. decorospora, N. limonensis and N. naponensis, which were collected from Central and South America. Natipusilla is characterized by small, superficial, light-colored, globose pseudothecia, fissitunicate asci, few or no pseudoparaphyses, and hyaline, one-septate to tardily two- or three-septate ascospores with or without a gelatinous sheath. The three Natipusilla species form a well supported clade, but their relationship to other members of the Dothideomycetes remains unclear.