Scombrids are the important component of pelagic fishery resources which include 54 species under 15 genera commonly known as mackerels, bonitos, and tunas. Due to the high commercial value attained, there are real chances of fraudulent substitution by species of inferior value. DNA based species identification methods can be applied to detect product adulteration, as well as to better contribute to the conservation and management of these species by providing accurate species identification independently of the age of the individuals or the tissue processed. In this study, a total of 15 commercially important scombrid species from Indian waters were analyzed. Due to the inadequacy of mitochondrial COI barcoding gene in discriminating between some Thunnus species, cytochrome b sequences were used instead. For all the 15 species, we propose a DNA character-based keys which uses a diagnostic combination of nucleotides and respective probes, including the first character-based keys and probes to differentiate between Thunnus albacares and T. obsesus.