The rice frog, Fejervarya multistriata, is an amphibian that is widely distributed in Guizhou Province, China. In this study, we determined the diversity and phylogeography of 10 geographic populations (252 individuals) from Tongren, Guizhou Province using 516-bp sequences of the mitochondrial D-Loop region. In total, 63 polymorphic sites, including four single-nucleotide polymorphisms and 59 parsimony-informative sites, and 18 haplotypes were detected. The number of haplotypes within populations ranged from one (WS and DJ) to six (JK). Haplotype diversity ranged from 0.000 (DJ and WS) to 0.793 (JK). The largest amount of nucleotide diversity was found in the SQ population (0.03111 ), whereas the smallest amount was in the DJ and WS (0.00000) populations. Phylogenetic analysis revealed that these 10 populations could be separated into two clusters: the first included CD, WS, YP, JK, and ST, and the second included DJ, YJ, YH, SQ, and SN. However, there was no significant divergence between populations based on pair-wise differences (FST). Overall, the 10 populations were divided into two subgroups and two D-Loop haplotype clusters. This study showed that the different rice frog populations in Tongren had low genetic diversity and little genetic flow, which has led to substantial differences in genetic divergence between western and eastern populations. These results show that geographical isolation, especially by mountains, has played an important role in limiting rice frog migration.