Abstract: Sequences of mitochondrial 16S rRNA, COX1 and Cytb genes were determined to establish the phylogenetic relationship among 11 species of anemonefish using the neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian methods with the divergence and genetic distances determined as well. The 3 genes had a combined length of 4 388 bp that consisted of 1 028 polymorphic and 616 parsimonious-information sites. The ratio of transition to transversion was 2.12, and the average A+T content was 54.7%, which was higher than 45.3% for C+G in the genes. It was found that (1) the subfamily Amphiprioninae was monophyly, but the genus Amphiprion was not monophyletic, implying Premnas should be reclassified; (2) the phylogenetic trees grouped the percula complex into a clade that formed the ancestral taxon of anemonefish, but the phylogenetic relationship was uncertain in regard to Premnas biaculeatus; and, while the Bayesian tree placed the percula complex and P. biaculeatus in a same clade, NJ and ML tree classified P. biaculeatus in a clade with subgenera Amphiprion, Paramphiprion and Phalerebus; and, (3) the genetic distance between species suggested a possibility of P. biaculeatus and Premnas epigrammata being identical.