Scombridae is a family of pelagic marine fishes that comprises 16 genera and 51 species. This family has been of significant commercial importance throughout history; however, the phylogenetic relationships within the Scombridae have been disputed due to the unclear taxonomic boundaries of the suborder Scombroidei, which includes six families, including Scombridae. Despite this, only a limited number of studies have been conducted on the Scombridae. In our study, eight species covering five genera of the Scombridae were selected, and one nuclear (ITS) and three mitochondrial DNA markers (CO1, Cytb, and D-loop) were used to amplify gene fragments. Additionally, we included homologous sequences from other Scombridae fishes obtained from GenBank. Our analysis constructed phylogenetic relationships of 48 Scombridae species in 14 genera. The results demonstrated that the three phylogenetic trees (NJ, ML, and BI) exhibited similar topologies, containing three major clades. One major clade indicated that Grammatorcynus bilineatus and Grammatorcynus bicarinatus did not cluster with other species in the Scombridae; another clade comprised the genera Scomber and Rastrelliger ; the third clade consists of the remaining Scombridae species. Notably, the study showed that Gasterochismatinae and Scombrinae were not sister groups; Allothunnus (tribe Thunnini) and Cybiosarda (tribe Sardini) clustered into a clade, suggesting that Sardini and Thunnini were non-monophyletic. Overall, this research enhances the understanding of phylogenetic relationships within the Scombridae and provides basic information to aid further research.