The Myctophidae family is widely distributed in mesopelagic oceans worldwide and plays a significant role in the pelagic food chain due to its vertical migratory behavior. Myctophidae are diverse and the morphological differences between genera are not obvious. Therefore, the use of molecular markers for Myctophidae identification would be advantageous. In this study, we amplified 56 Myctophidae samples collected from the Northwest Pacific using the Cyt b and 12S rRNA genes. These amplified sequences were subsequently used for phylogenetic analyses alongside sequences from 29 other Myctophidae species in 18 genera from the GenBank database. We explored the applicability of Cyt b and 12S rRNA genes for identifying Myctophidae species. The results indicate that the 56 samples belonged to 6 species: Ceratoscopelus warmingii, Symbolophorus californiensis, Myctophum asperum, Tarletonbeania crenularis, Notoscopelus japonicus, and Notoscopelus sp. The intra-specific and inter-specific genetic distances based on the Cyt b gene were more pronounced than those based on the 12S rRNA gene. The difference between the average inter-specific and intra-specific genetic distances of Cyt b and 12S rRNA gene was 25 and 26, respectively. These results indicated that both genes meet the basic requirements for DNA barcodes. Phylogenetic analysis evidenced that the six Myctophidae species each formed an independent clade based on Cyt b and 12S rRNA gene sequences. However, in the evolutionary tree constructed based on the Cyt b gene, each fish clustered more closely with sequences in the GenBank database. In summary, both Cyt b and 12S rRNA genes could be used as DNA barcodes for identifying Myctophidae species, amd the Cyt b gene was particularly suitable for analyzing phylogenetic relationships.