We developed an individual identification method for goldfish based on morphological variation of the iris. Each goldfish has a few dark lines (eye marks) in the rostral and caudal portion of the iris, which are blood vessels underneath the silvery reflective layer. Through the blood vessels and the locally thin reflective layer, the pigment cell layer is partially visible as a dark line. The pattern of the blood vessels was found to be temporally stable and unique to each individual. Using this feature, we successfully identified 10 individual goldfish, each sampled three times within a 4-month time period. The eye mark identification method was confirmed for a further 20 goldfish by comparison with identification based on screening at polymorphic microsatellite loci. The eye mark method is 100% accurate and can be used for studies in which multiple observations of individually identified goldfish are needed over long time periods, to avoid invasive tagging or individual housing that may affect the behavior of the fish.