摘要:Abstract Aquatic invasive species, such as the signal crayfish (Pacifastacus leniusculus), present a major threat to freshwater ecosystems. However, these species can be challenging to detect in recently invaded habitats. Environmental DNA (eDNA)–based methods are highly sensitive and capable of detecting just a few copies of target DNA from non‐invasively collected samples. Therefore, they have considerable potential for broad‐scale use in mapping and monitoring the spread of invasive species. In this study, we aimed to increase our understanding of the current distribution of signal crayfish in a headwater stream system in the United Kingdom (tributaries of the River Wharfe, Addingham, Yorkshire). Environmental DNA sampling, assessment of water chemistry variables, and conventional crayfish hand‐searching were conducted across 19 study sites in five tributary streams. Using hand‐searching, we detected signal crayfish at 26% of the sites (5/19 study sites). However, using eDNA‐based methods, occupancy increased to 47% of study sites (9/19). Our sampling revealed previously unknown sites of crayfish occupancy, and using eDNA‐based methods, we were able to define the geographical extent of the invasion front in each headwater stream sampled. This study highlights that eDNA‐based methods are well‐suited for detecting newly established signal crayfish populations in recently invaded habitats, even when the invasive species is at low abundance and, therefore, might otherwise be under‐represented or undetected using conventional survey methods. Our study provides further evidence that headwater stream ecosystems are particularly vulnerable to signal crayfish invasion. However, their geomorphological features may make methods used to reduce or prevent invasive crayfish dispersal more effective than in other freshwater ecosystems.