Abstract: This study examined the age structure of the Loach, Homatula pycnolepis through the otolith growth rings in 204 individual specimens collected from the Xiaomengtong River of the Nujiang River (Salween River) basin in April, 2008. There were only two different age classes, 1 and 2 years of age—no 3 year olds were detected. The age structure of H. pycnolepis was simple. The complete mitochondrial DNA cytochrome b gene sequences (1140) of 80 individuals from 4 populations collected in the Nujiang River drainage were sequenced and a total of 44 variable sites were found among 4 different haplotypes. The global haplotype diversity (Hd) and nucleotide diversity (Pi) were calculated at 0.7595, 0.0151 respectively, and 0, 0 in each population, indicating a consistent lack of genetic diversity in each small population. There was obvious geographic structure in both the Nujiang River basin (NJB) group, and the Nanding River (NDR) group. The genetic distance between NJB and NDR was calculated at 0.0356, suggesting that genetic divergence resulted from long-term isolation of individual population. Such a simple age structure and a lack of genetic diversity in H. pycnolepis may potentially be due to small populations and locale fishing pressures. Accordingly, the results of this study prompt us to recommend that the NJB, NDR and Lancang River populations should be protected as three different evolutionary significant units or separated management units.