The Hong Kong newt (Paramesotriton hongkongensis) has a restricted distribution in Hong Kong and its adjacent coastal area in China. We employed amplified fragment length polymorphism (AFLP) DNA markers to examine the genetic diversity and differentiation of P. hongkongensis populations (101 individuals) from three streams. Two mainland populations are separated by Tai Mo Shan, the highest peak in Hong Kong. On Hong Kong Island, there is only one population. We investigated whether the marine barrier would affect the population genetic structure and diversification of Hong Kong newts more than an upland peak barrier. With seven selected primer combinations, 476 AFLP markers with 102 polymorphic loci indicated that newt populations exhibited considerable genetic differentiation. Our results indicate that the two mainland populations, separated by the mountain ridge, were genetically more distant from each other than from the island population separated by the sea barrier that was created about 6000 years ago. The terrestrial biogeographic barrier appears to be crucial in influencing genetic divergence of Hong Kong newt populations.