Anemonefishes (genus Amphiprion) are one of the most popular marine ornamental fish among aquarists and also breed under captive condition with relatively little effort compared to other marine fish species. Though they are known to socially controlled, protandrous sex changing fish, when two immature anemonefish with ambisexal gonads are raised together in captivity, the two differentiate into a male and a female directly and form a breeding pair. However, not all the formed pairs reach to spawn, and there is a possibility that partner preference may exist in anemonefishes. It seems contrary to reason, because the social group of anemonefish formed totally by accident in the wild, then existence of partner preference must disturb their breeding success. So in this study, we evaluated behavioral and physiological differences between the “breeding” (B) and “non-breeding” (NB) pairs of false clown anemonefish Amphiprion ocellaris. Behavioral observation revealed that NB females monopolized the shelter, whereas B females allowed R males to use the shelter and stayed together in the shelter with frequency. Both NB and B females possessed mature ovarian tissue, but in contrast NB males had smaller amount of testicular tissue and lower plasma 11-ketotestosterone levels compared to B males. Transcription levels of genes encoding gonadal steroid-metabolizing enzymes (cyp19a1a and 11β-HSD) and brain nonapeptides receptors (AVTRs and ITR) did not differ between the experimental groups, rather to show sex difference. These result suggested that partner preference exist in the pairs of A. ocellaris, but brain nonapeptide may not involve in pair bonding. Females may have the initiative in spawning and may suppress sexual maturity of unacceptable males in some way.