Typhlomys is a relict genus of Platacanthomyidae. Typhlomys spp. are mainly distributed in the mountainous forests of southern China and northmost Vietnam. Although four species and one putative new species were recognized in a previous study, their karyotypes remain undetermined. In addition, a revision of taxonomic positions of several geographic populations were not determined. In the current study, we determined the karyotypes of Typhlomys cinereus and Typhlomys daloushanensis for the first time, and conducted molecular phylogenetic analysis to clarify the taxonomy and distribution of Typhlomys spp. in China. The diploid number of chromosomes in T. cinereus is 2n=36, with a karyotype of 14(M, SM) + 20 (A), XY (SM, A), and 6 Ag-NORs. T. daloushanensis has 2n=56, 2 (SM) + 52 (A), XX (SM, SM), and 4 Ag-NORs karyotypes. These karyotypes are distinct from that of T. chapensis (2n=38) from Vietnam and southern Yunnan, supporting the validity of each species. Phylogenetic analysis using three mitochondrial genes (Cyt b, ND2 and COI), suggested the Mts. Qinling and Nanling populations should be assigned to T. daloushanensis and T. cinereus, respectively. In addition, the populations from southern Yunnan and southern Guizhou represent distinct evolutionary lineages, implying unrecognized taxa may exist in the Karst mountains in Guangxi, Guizhou and Yunnan.