Yan-Horn Lee, Tsair-Bor Yen, Chiu-Fen Chen, and Mei-Chen Tseng (2018) Thunnus tunas in Scombridae are divided into the temperate subgenus Thunnus (bluefin group) and tropical subgenus Neothunnus (yellowfin group) species based on anatomic traits and distributions. The main purpose of this study was to examine the systematic status of T. obesus based on karyotype, cytochrome (Cyt) b gene, and 5S ribosomal DNA sequences. All T. obesus, T. albacares, T. alalunga, and T. orientalis specimens were caught in southeastern coastal waters off the main island of Taiwan. The karyotypical formula of T. obesus was 2 m + 2 st + 44 t, that of T. albacares was 2 m + 2 sm + 2 st + 42 t, that of T. alalunga was 2 m + 2 sm + 2 st + 42 t, and that of T. orientalis was 2 m + 2 sm + 44 t (m: metacentric; sm: submetacentric; st: subtelocentric; t: telocentric chromosome). According to a molecular genetics analysis for these species using Cyt b gene sequences (1141 bp), interspecific genetic distances ranged from 0.004 (T. orientalis vs. T. alalunga) to 0.038 (T. alalunga vs. T. obesus). The genealogy tree exhibited these 4 species as being categorized into 4 monophyletic groups with high bootstrapping values; T. alalunga and T. orientalis are sister species. This result suggests that the species currently allocated in Thunnus and Neothunnus might need new taxonomic characters to redefine the monophyly of the two subgenera. The sequence lengths of all cloned 5S genes from the 4 species ranged from 327-342 bp. Interspecific genetic distances ranged from 0.016 (T. orientalis vs. T. alalunga) to 0.111 (T. orientalis vs. T. albacares). The phylogenetic tree based on 5S rDNA shows T. obesus divided into 2 groups: one similar to T. albacares and the other close to T. orientalis. These results imply that Thunnus tunas have a common synapomorphic character with Scombridae fish (2n = 48) and high numbers of telocentric chromosomes (42-44). Thunnus orientalis and T. alalunga are sister based on molecular data. Thunnus obesus may have been derived from a more-complicated speciation processes.