Environmental DNA (eDNA) is frequently used for detecting species and describing biodiversity through metabarcoding techniques. More recently, there has been emerging evidence that eDNA can be used to investigate intraspecific variability, providing novel pathways to explore population genetics questions. However, it can be difficult to distinguish between true intraspecific variation and PCR/sequence error, and the presence of DNA from multiple individuals makes using traditional frequency‐based approaches challenging. Here, we explore how eDNA can be used to investigate population structure of Hector's dolphin (Cephalorhynchus hectori), an endemic, endangered, and culturally important (taonga) species. In doing so, we present a simple and effective method to filter out noise due to PCR/sequence error and show how treating haplotype detections equally can provide similar results to frequency‐based approaches from traditional sampling methods. Over the 2022/23 Austral summer, we collected 85 water samples close to Hector's dolphins, and three negative controls, across three areas on the east coast of Aotearoa New Zealand's South Island: Banks Peninsula (n = 41), Timaru (n = 33), and Dunedin (n = 14). We targeted a 348 bp region of the cetacean D‐loop in the mitochondrial DNA (mtDNA) and obtained positive detections in 68 (77%) water samples, confidently identifying seven haplotypes across the study area. The occurrence of specific haplotypes and the overall frequencies in Banks Peninsula and Timaru matched well with previous tissue‐based studies and were similar to other East Coast South Island (ECSI) subpopulations. In Dunedin, however, our results indicate a closer relationship to South Coast populations, suggesting that the membership within the ECSI population be reconsidered, which has implications for how this subpopulation is managed. We show that eDNA sampling can be used to elucidate matrilineal population structure for Hector's dolphin and provide a simple method that could be applied to other eDNA‐based studies of any taxa.
