This study documents the morphology and phylogeny of ascomycetes collected from karst landscapes of Guizhou Province, China. Based on morphological characteristics in conjunction with DNA sequence data, 70 species are identified and distributed in two classes (Dothideomycetes and Sordariomycetes), 16 orders, 41 families and 60 genera. One order Planisphaeriales, four families Leptosphaerioidaceae, Neoleptosporellaceae, Planisphaeriaceae and Profundisphaeriaceae, ten genera Conicosphaeria, Karstiomyces, Leptosphaerioides, Neoceratosphaeria, Neodiaporthe, Neodictyospora, Planisphaeria, Profundisphaeria, Stellatus and Truncatascus, and 34 species (Amphisphaeria karsti, Anteaglonium hydei, Atractospora terrestris, Conicosphaeria vaginatispora, Corylicola hydei, Diaporthe cylindriformispora, Dictyosporium karsti, Hysterobrevium karsti, Karstiomyces guizhouensis, Leptosphaerioides guizhouensis, Lophiotrema karsti, Murispora hydei, Muyocopron karsti, Neoaquastroma guizhouense, Neoceratosphaeria karsti, Neodiaporthe reniformispora, Neodictyospora karsti, Neoheleiosa guizhouensis, Neoleptosporella fusiformispora, Neoophiobolus filiformisporum, Ophioceras guizhouensis, Ophiosphaerella karsti, Paraeutypella longiasca, Paraeutypella karsti, Patellaria guizhouensis, Planisphaeria karsti, Planisphaeria reniformispora, Poaceascoma herbaceum, Profundisphaeria fusiformispora, Pseudocoleophoma guizhouensis, Pseudopolyplosphaeria guizhouensis, Stellatus guizhouensis, Sulcatispora karsti and Truncatascus microsporus) are introduced as new to science. Moreover, 13 new geographical records for China are also reported, which are Acrocalymma medicaginis, Annulohypoxylon thailandicum, Astrosphaeriella bambusae, Diaporthe novem, Hypoxylon rubiginosum, Ophiosphaerella agrostidis, Ophiosphaerella chiangraiensis, Patellaria atrata, Polyplosphaeria fusca, Psiloglonium macrosporum, Sarimanas shirakamiense, Thyridaria broussonetiae and Tremateia chromolaenae. Additionally, the family Eriomycetaceae was resurrected as a non-lichenized family and accommodated within Monoblastiales. Detailed descriptions and illustrations of all these taxa are provided.