The abundance of many marine mammals is declining due to local, regional, and global climate stressors that characterize the Anthropocene. Long-term monitoring is crucial for understanding how these declining populations respond to further environmental stress, and developing non-invasive genetic sampling strategies is needed to guide their recovery effectively. This study tested if complete mitochondrial genomes can be assembled from environmental DNA (eDNA) metagenomics scat samples taken non-invasively using the South American Sea Lion Otaria byronia as a model and examined if the retrieved mitochondrial genomes can facilitate non-intrusive population genetic studies. Complete mitochondrial genomes of O. byronia were assembled from each of a total of 30 eDNA samples with coverages greater than 40× using a target-restricted-assembly bioinformatics strategy. The AT-rich mitochondrial genomes contained 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. An Analysis of Molecular Variance and pairwise ΦST tests demonstrated statistically significant genetic dissimilarity among the sampled populations in the Pacific and Atlantic basins.