Tunas are commercially important fishery worldwide. There are at least 13 species of tuna belonging to three genera, out of which genus Thunnus has maximum eight species. On the basis of their availability, they can be characterised as oceanic such as (longtail tuna). Although these two are different species, morphological differentiation can only be seen in mature individuals, hence misidentification may result in erroneous data set, which ultimately affect conservation strategies. The mitochondrial DNA cytochrome oxidase c subunit I (COI) gene is one of the most popular markers for population genetic and phylogeographic studies across the animal kingdom. The present study aims to study the sequence conservation and variation in mitochondrial Cytochrome Oxidase I (COI) between these two species of tuna. COI sequence analysis of yellowfin and longtail revealed the close relationship between them in Thunnus genera. The present study is the first direct comparison of mitochondrial COI sequences of these two tuna species.