Genetic diversity represents the evolutionary potential of a population, and allows insights into its demographic history and genetic structure; understanding these aspects of a population is crucial for conservation planning. The Neotropical otter (Lontra longicaudis) is distributed from Argentina to Northern Mexico and is currently listed as ‘data deficient' by the International Union for the Conservation of Nature (IUCN). To contribute to conservation planning for the species, we used non-invasive sampling to obtain mtDNA control region sequences from 44 individuals from across Mexico. We examined country-wide genetic diversity and demographic history, as well as genetic structure among 3 regions: North Pacific, South Pacific, and Atlantic. Haplotypes identified in Mexico were combined with Central and South American haplotypes in order to examine phylogeographic patterns and identify evolutionary significant units (ESUs). Results show lower genetic diversity in Mexico compared to recent estimates for South American populations. Analyses of demographic history in Mexico indicated an expansion coinciding with climatic changes at the end of the Pleistocene. Genetic structure was high among North Pacific/ South Pacific (F_ST = 0.48) and North Pacific/Atlantic (F_ST = 0.49), potentially due to mountain chains acting as barriers to female gene flow among these regions. On the other hand, we identified a potential corridor for gene flow among South Pacific and Atlantic. Phylogeographic analyses identified a distinct lineage distributed in North and Central America. We propose this represents a distinctive ESU which should be considered for separate conservation management.