Athyonidium chilensis is a sea cucumber species that inhabits both the intertidal and subtidal zones of the southeast coast of the Pacific Ocean. This species is considered a commercially valuable fishery resource in Peru and Chile, which is exported to Asian countries for human consumption as a dried product. Despite its commercial importance, the spatial distribution of the genetic variability in A. chilensis along its geographic range remains unstudied. This information is crucial to define population stocks and to establish science-based management and conservation plans. Hence, our study aimed to assess the genetic structure and diversity of A. chilensis using DNA from adult specimens from six natural banks located between 18 °S and 42 °S. We analyzed 364 sequences from two genes (241 COI sequences and 123 ATPS-b sequences), which not only revealed high genetic diversity among A. chilensis populations from Chile but potentially also the presence of a second, cryptic species. Additionally, significant genetic distances were found among locations, with genetic flow decreasing with increasing spatial distance, suggesting different population stocks along the southeast coast of the Pacific Ocean. We provide crucial, baseline information to move forward with management plans for the preservation of genetic diversity for the species and the fishery to persist.