Abstract: The genus Cranoglanis is a group of fish only distributed in East Asia. They live in the drainages of Guangdong, Guangxi,Yunnan and Haiuan Province, China, and the Red River drainage in northern Vietnam. At present,the wild re-source of Cranoglanis bouderius has decreased rapidly because of excess utilized, and there are still some arguments on the validity of the species of the genus Cranoglanis. In order to determining the genetic structure and the validity of the spe-cies, AFLP technique was used to analyze the genetic diversity of Pearl River C. bouderias and Hainan C. bouderius. Sixty individuals from the two populations (thirty individuals per population) were analyzed by using eighteen primer combina-tions. The amplified bands of the eighteen primer combinations were in focus and repeated. The models of amplified band had distinct difference. The result of study showed that both populations had high percent of polymorphic loci and number of special bands. The genetic similarities was 0.9462?.0237 and 0.9465?.0226 within the population of Pearl River C. bouderius and the population of Hainan C. bouderius respectively, and was 0.9367?.0231 between Pearl River C. bouderius and Hainan C. bouderius, the genetic similarities between populations was lower than that within population. The genetic distance was 0.0634?.0230 between Pearl River C. bouderius and Hainan C. bouderius. The analyzed of phylo-genetic trees of two populations suggested that individuals of the same population combined together at first, and then two populations combined. The preliminary analysis result indicates that there are no prominent differences between genetic structure of Pearl River C. bouderius and Hainan C. bouderius, so they should be considered as one species.