This study investigates the genetic diversity and population structure of the greasyback shrimp, Metapenaeus ensis (De Haan, 1844), within the Tam Giang – Cau Hai lagoon, Vietnam, by analyzing mtCOI genes from 91 individuals collected across four populations in nine sampling locations. High genetic diversity was found, with 34 unique haplotypes and 38 genetic variations identified. Most genetic variation occurred within populations (AMOVA), suggesting high gene flow. Low and non-significant Fst values and close genetic distances confirmed minimal differentiation among populations. These findings provide the first insights into M. ensis population genetics in this lagoon, informing conservation and management efforts. Understanding genetic diversity is crucial for sustainable management and conservation of marine species. This research aids in understanding population resilience, potential impacts of overfishing, and may assist in developing sustainable harvest strategies of this economically important shrimp species.