Spring-fed ecosystems of the US Great Basin are home to an extraordinary diversity of endemic and/or threatened aquatic organisms. Aquatic organisms continuously shed DNA into their surroundings. This “environmental DNA (eDNA)” is detectable quantitative Polymerase Chain Reaction (qPCR) and metabarcoding. Here we report on optimized sampling and DNA extraction protocols and validation of published and custom molecular probes utilizing mitogenome targets for endemic and invasive species in spring-fed environments of the Mojave Desert. Published Metabarcoding fish primers (e.g. MiFish (12S rRNA gene)) were used to document an ongoing mosquitofish (Gambusia affinis) invasion at the Shoshone pupfish refugium (Cyprinodon nevadensis shoshone) in Shoshone, CA. This assay was also used to verify known and cryptic populations of Amargosa speckled dace (Rhinichthys osculus navadensis) in Amargosa River headwater springs and explore diversity of endemic and invasive fish assemblages on the Muddy and Virgin Rivers. The river work established many species, but some highlights include the detection of strong populations of Virgin River Chub (Gila seminuda) in the Muddy and Virgin Rivers and chub and speckled dace (Rinichthys osculus) in the Virgin. A rogue’s gallery of invasives was also present in both, especially the Virgin River below Mesquite, NV, where red shiner (Cyprinella lutrensis)), common carp (Cyprinus carpio) and channel catfish (Ictalurus punctatus) dominated. Conversely, blue tilapia (Oreochromis aureus), which have been eradicated from the Muddy and have been detected sporadically in the middle Virgin, were not detected, although they were easily detected in the Las Vegas Wash. Specialty assays included a sensitive and specific qPCR assay for Moapa dace (Moapa coriacea) and new qPCR and metabarcoding primers for super-invasive red swamp crayfish (Procambarus clarkii), which have been tested at Ash Meadows, NV.