The genera Kernia and Acaulium comprise species commonly isolated from dung, soil, decaying meat and skin of animal. The taxonomy of these fungi has been controversial and relies mainly on morphological criteria. With the aim to clarify the taxonomy and phylogeny of these fungi, we studied all the available ex-type strains of a large set of species by means of morphological and molecular phylogenetic analyses. Phylogenetic analysis of the partial internal transcribed spacer region (ITS) and the partial 28S rDNA (LSU) showed that the genera Kernia and Acaulium were found to be separated in two distinct lineages in Microascaceae. Based on morphological characters and multilocus phylogenetic analysis of the ITS, LSU, translation elongation factor 1α and β-tubulin genes, the species in Kernia and Acaulium were well separated and two new combinations are introduced, i.e. Acaulium peruvianum and Acaulium retardatum, a new species of Kernia is described, namely Kernia anthracina. Descriptions of the phenotypic features and molecular phylogeny for identification are discussed for accepted species in two genera in this study.