Chub mackerel (Scomber japonicus) and blue mackerel (Scomber australasicus) are important commercial fishes around the coastal waters of Japan. In this study, a novel polymerase chain reaction (PCR)-based species identification method was developed and used to discriminate between these two species and their hybrids. In this PCR method, a species-specific insertion or deletion marker is used to enable the amplification of the same locus in two Scomber species with one set of primers. The size difference in the amplified DNA fragment was approximately 300 bp, and the fragments could be separated using standard agarose gel electrophoresis. To validate the effectiveness of the method, the two mackerel species were sampled in various areas of Japan and identified using PCR. In 208 chub mackerel and 118 blue mackerel, PCR-based identification was 100% consistent with external morphology-based species identification. Moreover, in DNA samples of F1 hybrid larvae obtained via artificial insemination of the two mackerel species, hybrids were identified with 100% accuracy using the PCR method. Chub mackerel, blue mackerel, and their hybrids are typically difficult to identify by their morphological characteristics. Thus, our PCR method will help in identifying these fishes and will contribute to assessing mackerel stocks in Japan.