A key component to conservation is an accurate understanding of genetic subdivision within a species. Despite their ecological and economic importance, relatively little is understood about the genetic structuring of Yellowstone cutthroat trout in Yellowstone National Park. Here, we use traditional (Fst, Rst, Nm, and AMOVA) and modern (Bayesian assignment tests, coalescent theory, and nested clade analysis) analytical approaches to describe the population genetic subdivision of cutthroat trout spawning populations in Yellowstone Lake and to identify genetically distinct population segments throughout Yellowstone National Park. Evidence for restricted gene flow between spawning populations within Yellowstone Lake was detected using nested clade analysis. This is the first molecular evidence for restricted gene flow between spawning populations in Yellowstone Lake. In contrast, traditional methods such as Fst and Rst as well as the Bayesian clustering program STRUCTURE v2.0 failed to detect evidence for restricted gene flow. Across our sampling range within Yellowstone National Park, eleven genetically distinct cutthroat trout population segments were detected. These showed a general pattern of small, isolated populations with low genetic diversity in headwater streams and wide-spread, genetically diverse populations in higher-order rivers. We recommend populations be managed to maintain current levels of genetic diversity and gene flow. Based on the recent decline of and distinct morphological, behavioral, and genetic nature of cutthroat trout in Yellowstone Lake, we recommend the Yellowstone Lake spawning populations collectively be recognized as an evolutionarily significant unit.