The three mackerel species, Rastrelliger kanagurta, Rastrelliger brachysoma, and Rastrelliger faughni, co-occur in the central part of the Indo-West Pacific Ocean, and together represent important fisheries resources. However, they have sometimes been collectively referred to as “Rastrelliger spp.” in the fisheries statistics, owing to their morphological similarity. Therefore, species-specific data on landings, effort, and population status are lacking, undermining the appropriate resource management. The present study provides a rapid and cost-effective method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis to distinguish the three species. Digestion of the PCR products of 1247 base pairs of mitochondrial DNA encompassing the complete sequence of cytochrome b gene using two restriction enzymes yielded diagnostic restriction patterns for the three species, thereby enabling accurate identification.