The Lower Colorado River Basin ecosystem plays host to dozens of nonnative fishes. Within Grand Canyon National Park, nonnative species such as smallmouth bass, rainbow trout, brown trout, flathead catfish and more often co-occur with federally threatened or endangered species, including the native and endemic humpback chub and razorback sucker. Understanding the genetic diversity of fish within the ecosystem is a vital tool for monitoring and conserving these species. We used environmental DNA (eDNA) as a non-invasive method for sampling the 12S mitochondrial region of fish genomes within the Lower Colorado River Basin (LCRB) at 44 unique sites, including the mainstem Colorado River, major tributaries, and tailwaters directly below dams. We isolated eDNA from these water samples and amplified the products via polymerase chain reaction (PCR) and the MiFish-U metabarcoding primer. Amplicons were then sequenced and sorted into operational taxonomic units (OTUs). We found that native fishes (N=4) harbored an average of 16 distinct 12S OTUs (range: 8 to 22), while nonnative fish species (N=26) had an average of 2 distinct 12S OTUs (range: 1 to 6). Mainstem and tributary sites contained three times as many nonnative fish species as native ones, but these nonnative fish have, on average, one-sixth the genetic diversity of native species. Tailwaters directly below dams were dominated by genetically poor nonnative species. The Colorado River is a highly modified system where native fishes, such as humpback chub, have undergone historic genetic bottle necks; the retention of high genetic diversity despite these challenges indicates the persistence of these species within microhabitats. Exploring the mechanisms behind the retention of this high genetic diversity would inform fish conservation efforts.