Abstract: Background and Objective: Clarias batrachus is native to Asia, Sumatra was one of the islands that were divided due to the division of Sundaland because of a transgression event. The Bukit Barisan Mountains divide Sumatra into two parts: West and East. Therefore, it is important to research C. batrachus phylogeography. One of the mtDNA genes that can be used for phylogeographic is the CO1 gene. This study examined the association between geographic isolation in Sumatra's Eastern and Western watersheds and the phylogenetic relationships between C. batrachus populations in Sumatra and other Southeast Asia using the CO1 gene. Materials and Methods: Clarias batrachus samples were collected from five populations 7 (Mungo River, Sinamar River, Agam River, Anai River and Lubuak Paraku River) from West Sumatra. The method of this research used the molecular method. The DNA isolation based on the COI gene follows the Invitrogen PureLinkTM Genomic DNA Mini Kit protocol. Haplotype network using software Popart v.1.7 and molecular evolution analysis using software MEGA. Results: Based on a total of 28 sequences (including comparisons and outgroups) with 504 base pairs of the CO1 gene from C. batrachus, three haplogroups and 19 haplotypes were identified using the haplotype analysis. Speciation events between haplogroup 1 estimates are 0.41 Mya haplogroup 2 estimates are 0.50 Mya. Conclusion: The separation of Sundaland and the separation in Sumatra’s Eastern and Western watersheds affects the kinship relationship of these populations with variations in subspecies level.