Genetic diversity is one of the criteria commonly used for conservation status assessment. Panhai’s crocodile newts, Tylototriton panhai, is distributed in Phu Hin Rong Kla National Park (NP), where two major breeding pond sites have been reported: Head Quarter and Thap Boek. Phu Hin Rong Kla NP is a popular tourist destination and is easily accessible from main roads. In this study, genetic diversity and population genetic structure of T. panhai population at Phu Hin Rong Kla NP were examined based on mitochondrial NADH dehydrogenase 2 (ND2) sequences (n = 28, 1,035 bp) extracted from tail tissue samples obtained from Head Quarter (n = 15) and Thap Boek areas (n = 13). Head Quarter population revealed relatively higher genetic diversity indices, in terms of number of haplotype, haplotype diversity, and nucleotide diversity (N = 6, h = 0.7714, and π = 0.001785) compared to Thap Boek population (N = 3, h = 0.6538, and π = 0.000743). Although, the results of pairwise genetic distance (p-distance) ranged from 0-0.58%, F_ST values showed significant genetic differentiations (F_ST = 0.23472, P < 0.001) between Head Quarter and Thap Boek populations. Moreover, the median-joining haplotype network showed eight distinct haplotypes, including seven unique and one shared haplotypes. The results of this study suggested that gene flows between breeding ponds, which were 1.4 km. apart, were restricted and both populations were valuable as they contributed to overall genetic diversity and conservation of T. panhai at  Phu Hin Rong Kla NP.