AbstractThe Thunnini, or tuna, comprise many species with very different commercial values. The principal raw tuna product on the market is sashimi, for which the species used is difficult to identify through conventional morphological analysis. The present study amplified the cytochrome b gene (Cytb) of 4 major tuna species used for preparing sashimi—yellowfin tuna (Thunnus albacares), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), and Atlantic bluefin tuna (Thunnus thynnus)—and 4 species commonly mislabeled as components of tuna sashimi—albacore tuna (Thunnus alalunga), skipjack tuna (Katsuwonus pelamis), striped marlin (Tetrapturus audax), and swordfish (Xiphias gladius). Polymerase chain reaction (PCR) amplicons were digested with 5 restriction enzymes—Eco147 I, Hinf I, Mbo I, Xag I, and Hind II—to obtain characteristic restriction maps of the above‐mentioned raw tuna species and the commonly mislabeled species. An identification method using PCR restriction fragment length polymorphism (PCR‐RFLP) was established and validated using 39 commercial tuna sashimi samples, which verified that this method provides results consistent with those obtained by classical sequencing. PCR‐RFLP has several advantages over classical sequencing, such as simplicity, speed and accuracy. This technique could support species identification for raw tuna and sashimi.