Frigate tuna Auxis thazard thazard is a cosmopolitan species and one of the smallest members of the tribe Thunnini (the true tunas), and currently managed as a single stock. Genetic variation of this species was surveyed using sequence data of mitochondrial DNA D-loop region to test for the presence of genetic stock structure of frigate tuna along the Indian coast. A total of 364 individuals were sampled from 8 major fishing zones along the Indian coast. Significant genetic heterogeneity was observed for the sequence data (FST = 0.0439, P = less than 0.001). Analysis of molecular variance (AMOVA) showed significant genetic variation among the three groups analysed (FCT = 0.1223, P = less than 0.05), which was also supported by spatial AMOVA results. Therefore, the null hypothesis of single panmictic population of frigate tuna along the Indian coast can be rejected. Phylogenetic analysis of nucleotide sequences demonstrated that frigate tuna can be grouped into three different mitochondrial clades (Clades I, II and III). However, there were no significant genealogical branches or clusters of samples corresponding to sampling locality. The results suggest the possibility of three genetically differentiated units of frigate tuna across the coastal waters of India.