During our investigation of saprobic fungi in southwestern China, three micro-hyphomycetous fungi were isolated from dead wood in freshwater and terrestrial habitats in Guizhou Province. Phylogenetic analyses of ITS, LSU, and SSU sequences, performed using Maximum Likelihood and Bayesian Inference methods, confirmed that these isolates belong to Kirschsteiniothelia. Based on distinct morphological characteristics and molecular phylogenetic evidence, we describe three new species: Kirschsteiniothelia guizhouensis, K. weiningensis, and K. xishuiensis. Furthermore, the effectiveness of three DNA markers for species-level identification within Kirschsteiniothelia was evaluated using Assemble Species by Automatic Partitioning (ASAP) analysis, which identified the ITS nucleotide sequences as the most reliable marker for species differentiation within the genus.