Detection of environmental DNA (eDNA) has become a commonly used surveillance method for threatened or invasive vertebrates in both aquatic and terrestrial environments. However, use of eDNA methodologies for the detection of aquatic invertebrates (e.g., crayfish and insects) has been limited. Environmental DNA protocols can be especially useful for endangered invertebrates such as the Hine’s emerald dragonfly (Somatochlora hineana) where conservation efforts have been greatly hindered by the training, time, overall costs, and environmental impacts associated with conducting surveys in the calcareous fens occupied by this species. An essential step in developing such a protocol is to evaluate the dynamics of eDNA concentration under controlled and field conditions. In this study we examined the persistence and accumulation of eDNA from captive S. hineana larvae in experimental mesocosms at temperatures (5.0°C and 16.0°C) that reflect seasonal variation in their natural habitat, and we evaluated the usefulness of eDNA protocols for studying the distribution and abundance of invertebrates by assessing patterns of eDNA distribution for the Hine’s emerald dragonfly and its symbiont the devil crayfish, (Cambarus [=Lacunicambarus] diogenes) in the field over several months. In mesocosms, S. hineana eDNA persisted longer at 5.0°C but accumulated more readily at 16.0°C. In the field, life-history events affected seasonal variations in eDNA more significantly and consistently than temperature for both species. These data can be used to aid in conservation efforts for S. hineana and similar aquatic invertebrates.