Two new species of the genus Triplophysa from southwestern Guizhou Province, China, are described. These are Triplophysa ziyunensis Wu, Luo, Xiao & Zhou, sp. nov. and Triplophysa yaluwang Lan, Liu, Zhou & Zhou, sp. nov. from Maoying Town, Ziyun County, Guizhou Province, China. Triplophysa ziyunensis Wu, Luo, Xiao & Zhou, sp. nov. is distinguished from other hypogean species of the genus Triplophysa by having a combination of the following characteristics: body naked, scaleless, pigmented markings on surface of body, except ventral; eyes reduced, diameter 2.4–4.9% of head length; pelvic-fin tip extending to anus; tip of pectoral fin not reaching pelvic fin origin; anterior and posterior nostrils closely set, with anterior nostril elongated to a barbel-like tip; tip of outrostral barbel extending backward, not reaching anterior margin of the eye; lateral line complete; posterior chamber of air bladder degenerated; and fin differences. Triplophysa yaluwang Lan, Liu, Zhou & Zhou, sp. nov. is distinguished from other hypogean species of the genus Triplophysa by having a combination of the following characteristics: body naked, scaleless, with irregular pale and dark brownish brown markings, except ventrally; eyes reduced, diameter 4.6–6.1% of head length; pelvic-fin tip reaching anus; tip of pectoral fin not reaching to pelvic fin origin; anterior and posterior nostrils closely set, with anterior nostril elongated to a barbel-like tip; tip of outrostral barbel extending backward, not reaching to anterior margin of the eye; lateral line complete; posterior chamber of air bladder degenerated; and fin differences. Mitochondrial Cyt b revealed relatively small genetic differences, 1.4–2.0%, between the two new species and close relatives. Nuclear gene RAG1 indicated that the two new species possessed unique haplotypes with multiple linking mutations. This study emphasizes the importance of utilizing nuclear genes to identify new species in rapidly speciation cave species, with small genetic differences due to mitochondrial introgression occurring interspecies.