Climbing perch (Anabas testudineus) is one of the economically important freshwater fish in Aceh, Indonesia, but is threatened by habitat fragmentation, overfishing, and the introduction of alien fish species. Therefore, this study aims to analyze the genetic structure of A. testudineus populations in the inland waters of Aceh using the Cytochrome c Oxidase subunit I (COI) marker. A total of 173 samples representing 12 populations, namely Banda Aceh, Aceh Besar, Pidie, Pidie Jaya, Lhokseumawe, West Aceh, Southwest Aceh, East Aceh, Simeulue, South Aceh, Gayo Lues, and Southeast Aceh, were successfully sequenced with a homology level of 98.12-100%. The analysis produced 44 unique haplotypes, with haplotype diversity ranging from 0.476 to 0.857 (average 0.729). The fixation index (Fst) value of 0.770 (p<0.001) showed significant genetic fragmentation, with 77% of genetic variation attributable to population differentiation. The phylogenetic tree reconstruction formed 2 main clades, where clade I comprised the populations of Pidie, Banda Aceh, Aceh Besar, South Aceh, Lhokseumawe, and East Aceh. Meanwhile, clade II comprised the populations of Southwest Aceh, West Aceh, Simeulue, Pidie Jaya, Southeast Aceh, and Gayo Lues. These results confirmed limited gene flow between populations due to geographic isolation and anthropogenic pressures, showing the need for specific conservation approaches for each on unit.