Kawakawa Euthynnus affinis is an epipelagic migratory tuna species, widely distributed in the tropical and subtropical waters of the Indo-Pacific region. Kawakawa constitutes the largest tuna fishery in the Indian waters. In the present study, genetic variation was assessed using sequence analyses of Mitochondrial DNA (mtDNA) D-loop region. A 500 bp segment of D-loop region was sequenced in 400 samples collected from eight localities (Veraval (VE), Ratnagiri (RA), Kochi (KO), Kavaratti (KA), Port-Blair (PB), Tuticorin (TU), Pondicherry (PO), and Vizag (VI)) along the Indian coast. Analysis of molecular variance of mtDNA data revealed no significant genetic differentiation among sites the (phi = 0.0028, P = 0.20723) indicating a single population along the Indian coast. Phylogenetic analysis revealed no obvious phylogeographic pattern separating the eight samples of kawakawa. However, the genealogical relationships demonstrated that mtDNA D-loop sequences belong to two different clades (clade I and clade II). Clade I is the major clade which consists of more than 98% specimens from each regional population while clade II has individuals from only three populations (VE, PO, and VI). Results of genetic analyses of the present study support a single stock management of kawakawa along the Indian coast.