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The tire track eel, Mastacembelus favus, is an economically important freshwater fish in mainland Southeast Asia. Understanding the spatial population structure is important to design an effective and efficient management of the species. The aim of this study was to investigate the genetic diversity and structure of M. favus populations. In total, 519 individuals from 30 locations across its distribution range were genotyped using a set of eight polymorphic microsatellite loci. Results showed that populations in Mekong and Chao Phraya exhibited higher genetic variability than those from Gulf of Thailand and Malay–Thai Peninsula. Clustering results revealed two evolutionarily isolated groups geographically separated at Northeastern Cambodia; the more widespread group was further divided into two subclusters. The spatial genetic variation and structure of its populations are attributed to the evolutionary history and population dynamics of the species. The study provides important information for the management and conservation of M. favus in Southeast Asia. [ABSTRACT FROM AUTHOR]