Understanding the genetic diversity and population structure of anguillid eels is crucial for effective management. In this study, we used the mitochondrial DNA (mtDNA) D-loop marker to identify anguillid glass eels and assessed their genetic diversity across six sampling sites spanning three Southeast Asian countries. We also examined the population structures of Anguilla marmorata and A. celebesensis. We validated nine distinct species and subspecies of anguillid eels from 465 specimens collected in Indonesia (n=246), the Philippines (n=119), and Vietnam (n=100). High gene diversities were observed across the species, ranging from 0.979 (A. celebesensis) to 1.000 (A. bicolor bicolor, A. marmorata, and A. interioris). Correspondingly, nucleotide diversity values were relatively low, varying from 0.036 (A. marmorata) to 0.057 (A. interioris). A. marmorata (n=162) displayed a broad distribution, being observed across all six sites, with a significant yet low average fixation index of 0.05755. Pairwise comparisons of FST values between Cimandiri and other locations (except for Cagayan) were relatively high and statistically significant (p<0.01), indicating significant genetic differentiation of Cimandiri samples compared to most locations in this study. On the other hand, A. celebesensis (n=110), exhibited a non-significant and low fixation index (0.007), indicating no genetic differentiation in samples from Poso and Poigar Rivers, Indonesia. Our findings highlight the essential need for coordinated management strategies among regions that share common stocks. Additionally, this study underscores the efficacy of the mtDNA D-loop in precisely identifying glass eel species/subspecies and elucidating the genetic diversity and population structure of tropical anguillid eels.