The genus Karstsinnectes of blind fishes known as karst loaches from China was established in 2023 during a revision of the genus Oreonectes (Nemacheilidae). Six species are recorded in this study and some taxonomic progress has been made; however, the lack of genetic data for some species (e.g., K. anophthalmus) may have weakened our current understanding of Karstsinnectes. This study reassessed the phylogeny and evolutionary history of Karstsinnectes by integrating a combination of previously published and newly sequenced mitochondrial genomic data. A phylogenetic tree was developed that was able to divide Karstsinnectes into two clades corresponding to drainages and clarify the phylogenetic position of K. anophthalmus. Divergence times show that Karstsinnectes originated at the Oligocene/Miocene boundary (~22.37 Mya), with the most recent common ancestor occurring in the early Miocene (~18.87 Mya) and interspecific divergence occurring in the late Miocene. Ancestral area reconstruction suggests that the most recent common ancestor of Karstsinnectes most likely inhabited the Hongshui River basin and dispersed into the Zuojiang-Yujiang, Beipanjiang, and Youjiang river basins during the early Miocene (~18.87 Mya), middle Miocene (~12.78 Mya), and late Miocene (~6.71 Mya), respectively. The dispersal under the influence of orogenesis and a monsoon climate drove the speciation and diverse distribution of Karstsinnectes. Such findings are important for conservation considering that Karstsinnectes strictly inhabits deep caves. Additionally, the taxonomic status of the distributed Karstsinnectes population in Leiping Town, Daxin County, Guangxi, China was revised by combining genetic and morphological differences to describe this population as a new species, Karstsinnectes daxinensis Luo, Zhou & Zhou, sp. nov. The definition of the phylogenetic position of K. anophthalmus emphasizes the importance of using type locality material for the identification of cryptic species.
