Tenualosa ilisha is an important fish in the Indian Ocean regions and the most profitable fishery in Bangladesh. A total of 160 T. ilisha (∼250 gm) were collected from Chandpur (riverine), Noakhali (estuarine), Patuakhali and Cox's Bazar (marine). Besides, forty Hilsha kelee (outgroup) samples were collected from Bagerhat (estuarine) region of Bangladesh. Cytochrome b region of mtDNA of all samples were amplified by PCR. Forty-two PCR products of T. ilisha and six H. kelee were sequenced to investigate their genetic structures. A total of 305 polymorphic sites with 23 haplotypes were found in T. ilisha . A network of these haplotypes was constructed along with molecular differentiation of T. ilisha and H. kelee by phylogenetic analysis. High genetic variability (0.902) and low nucleotide diversity (0.193) were found among four populations. Analysis of molecular variance showed significantly different population structure (Fst 0.025, p < 0.001). Our study revealed a high genetic variability and distinct population structure in the T. ilisha populations. Moreover, the non-significant negative value of Tajima's D for Chandpur, Noakhali and Patuakhali populations and negative Fu (FS) value for Noakhali population suggested a recent population expansion of hilsa.