Freshwater fungi comprises a highly diverse group of organisms occurring in freshwater habitats throughout the world. During a survey of freshwater fungi on submerged wood in streams and lakes, a wide range of sexual and asexual species were collected mainly from karst regions in China and Thailand. Phylogenetic inferences using partial gene regions of LSU, ITS, SSU, TEF1α, and RPB2 sequences revealed that most of these fungi belonged to Dothideomycetes and Sordariomycetes and a few were related to Eurotiomycetes. Based on the morphology and multi-gene phylogeny, we introduce four new genera, viz. Aquabispora, Neocirrenalia, Ocellisimilis and Uvarisporella, and 47 new species, viz. Acrodictys chishuiensis, A. effusa, A. pyriformis, Actinocladium aquaticum, Annulatascus tratensis, Aquabispora setosa, Aqualignicola setosa, Aquimassariosphaeria vermiformis, Ceratosphaeria flava, Chaetosphaeria polygonalis, Conlarium muriforme, Digitodesmium chishuiense, Ellisembia aquirostrata, Fuscosporella atrobrunnea, Halobyssothecium aquifusiforme, H. caohaiense, Hongkongmyces aquisetosus, Kirschsteiniothelia dushanensis, Monilochaetes alsophilae, Mycoenterolobium macrosporum, Myrmecridium splendidum, Neohelicascus griseoflavus, Neohelicomyces denticulatus, Neohelicosporium fluviatile, Neokalmusia aquibrunnea, Neomassariosphaeria aquimucosa, Neomyrmecridium naviculare, Neospadicoides biseptata, Ocellisimilis clavata, Ophioceras thailandense, Paragaeumannomyces aquaticus, Phialoturbella aquilunata, Pleurohelicosporium hyalinum, Pseudodactylaria denticulata, P. longidenticulata, P. uniseptata, Pseudohalonectria aurantiaca, Rhamphoriopsis aquimicrospora, Setoseptoria bambusae, Shrungabeeja fluviatilis, Sporidesmium tratense, S. versicolor, Sporoschisma atroviride, Stanjehughesia aquatica, Thysanorea amniculi, Uvarisporella aquatica and Xylolentia aseptata, with an illustrated account, discussion of their taxonomic placement and comparison with morphological similar taxa. Seven new combinations are introduced, viz. Aquabispora grandispora (≡ Boerlagiomyces grandisporus), A. websteri (≡ Boerlagiomyces websteri), Ceratosphaeria suthepensis (≡ Pseudohalonectria suthepensis), Gamsomyces aquaticus (≡ Pseudobactrodesmium aquaticum), G. malabaricus (≡ Gangliostilbe malabarica), Neocirrenalia nigrospora (≡ Cirrenalia nigrospora), and Rhamphoriopsis glauca (≡ Chloridium glaucum). Ten new geographical records are reported in China and Thailand and nine species are first reported from freshwater habitats. Reference specimens are provided for Diplocladiella scalaroides and Neocirrenalia nigrospora (≡ Cirrenalia nigrospora). Systematic placement of the previously introduced genera Actinocladium, Aqualignicola, and Diplocladiella is first elucidated based on the reference specimens and new collections. Species recollected from China and Thailand are also described and illustrated. The overall trees of freshwater Dothideomycetes and Sordariomycetes collected in this study are provided respectively and genera or family/order trees are constructed for selected taxa.