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Otter populations are threatened by habitat loss, pollution, conflicts with humans, and illegal wildlife trade to meet the demand for pets, for their fur, and for parts used in traditional medicines. Baseline information on the distribution, population genetic diversity, and connectivity is crucial to inform conservation management decisions; however, reliable data from otter populations in Southeast Asia remain scarce. In this study, we conducted baseline otter fecal DNA surveys based on mitochondrial DNA (mtDNA) to identify species, assess the occurrence, and map the spatial distribution of genetic diversity and evolutionary relationships of otter populations using 1700 bp Cytochrome B ‐ Control Region and mitogenome from Nakai‐Nam Theun National Park in the Annamite Mountains of Lao PDR. Of the total 56 samples identified to species, the majority (87.5%) was of the widely distributed Eurasian otter with three haplotypes (Lutra lutra; LLLA01–LLLA03), with a calculated haplotype diversity of 0.600 and a nucleotide diversity of 0.00141 based on mitogenome. The second species was the Asian small‐clawed otter with only one haplotype detected (Aonyx cinereus; ACLA01). All Eurasian otter haplotypes were newly characterized and clustered within the strongly supported South–Southeast–North Asian clade of Lutra lutra. Compared with the European clade, the high mtDNA diversity of Lutra lutra in Nakai‐Nam Theun National Park potentially reflects long‐term demographic stability and lesser degree of population bottleneck during the last glacial maxima (LGM, ~21,000 years ago). The single haplotype detected in Asian small‐clawed otters had not been detected in previous genetic studies. Our research is the first otter‐specific noninvasive genetic study in Lao PDR and provides baseline insights into the otter population diversity in a regional priority site for biodiversity conservation. [ABSTRACT FROM AUTHOR]