Many species of coral reef fishes are distinguished by their colour patterns, but genetic studies have shown these are not always good predictors of genetic isolation and species boundaries. The genus Amphiprion comprises several species that have very similar colouration. Additionally, morphological characters are so variable, that sibling species can show a considerable overlap, making it difficult to differentiate them. In this study, we investigated the species boundaries between the sibling species pair A. ocellaris and A. percula (Subgenus Actinicola) and three closely related species of the subgenus Phalerebus (A. akallopisos, A. perideraion, A. sandaracinos) by phylogenetic analysis of mitochondrial cytochrome b and control region sequences. These two subgenera show strong differences in their patterns of species boundaries. Within the A. ocellaris/A. percula complex, five clades were found representing different geographic regions. Two major divergences both with genetic distances of 4–7% in cty b and 17–19% in the d-loop region indicate the presence of three instead of two deep evolutionary lineages. The species of the subgenus Phalerebus show three monophyletic clades, independent of the geographical location of origin, but concordant to the morphological species classification. The genetic distances between the Phalerebus species were 2–5% in cty b and 10–12% in the control region.