The loss and degradation of freshwater habitats in Canada threaten the rich array of plant and animal species supported within these ecosystems, threatening the biodiversity that these ecosystems sustain. Assessing the distribution of the species in these threatened ecosystems is necessary for the effective monitoring of the well-being of their populations and the ecosystem, but it can be difficult, as some species can evade traditional survey methods due to their elusiveness, size, range, or rarity. Environmental DNA (eDNA), which describes the pool of DNA present in an environment that can be collected in and isolated from environmental samples, offers an alternative to traditional surveying that can be more sensitive and less invasive. Therefore, I developed and validated TaqMan™ MGB probe-based eDNA assays for six freshwater fishes of conservation concern in Manitoba: Bigmouth Buffalo Ictiobus cyprinellus, Chestnut Lamprey Ichthyomyzon castaneus, Golden Redhorse Moxostoma erythrurum, Hornyhead Chub Nocomis biguttatus, Northern Brook Lamprey Ichthyomyzon fossor, and Silver Lamprey Ichthyomyzon unicuspis. The designed assays were validated in situ using paired eDNA and capture-based sampling and used to map the distribution of the above species from water samples collected and filtered from 129 sites across Manitoba in 2021–2023. I detected the eDNA of four of the target species outside of their historic range: Bigmouth Buffalo in the Roseau, Morris, and Assiniboine rivers; Golden Redhorse in Hazel Creek; Hornyhead Chub in waterbodies of the Winnipeg River watershed where it has not been reported previously; and Northern Brook Lamprey in Boggy River. Apart from Northern Brook Lamprey, the eDNA of all target species was not detected in at least one site within their historic range. Further research is needed to confirm if the lack of eDNA detections of the target species within their historic range is a result of range restriction, or if a refinement of the methods used is needed.