The binding position as well as the number of docosahexaenoic (DHA) moieties in tuna and in bonito orbital fat triglycerides (TG) were investigated. DHA was dominantly bound in the sn-2 position. The most abundant TG molecular species was 1 (or 3)-palmitoy1-2, 3 (or 1)-di-doosahexaenoin. This molecular species was at most less than 8% in TG fraction. DHA was distributed to many kinds of molecular species, mostly in triacid type and diacid TGs. Tridocosahexaenoin was present in a trace amount. When tuna and bonito orbital fats were each randomized, both TGs could be easily discerned by their molecular species pattern. It was concluded that the basic TG molecular species pattern of bonito orbital fat matches that of the tuna, and that these can be substituted for each other as a valuable resource.