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Piute ground squirrels (Spermophilus mollis) are distributed continuously in habitat dominated by native shrubs and perennial grasses in the Snake River Birds of Prey National Conservation Area in Idaho, U.S.A. This habitat is being fragmented and replaced by exotic annual plants, changing it to a wildfire-dominated system that provides poor habitat for ground squirrels. To assess potential effects of this fragmentation on ground squirrel populations, we combined an estimate of effective population size (Ne) based upon a demographic study with a population genetic analysis. The study area included three subpopulations separated from each other by 8–13 km. The ratio of effective population size to census number (Ne/N) was 0.57. Combining Ne/N with dispersal distances from a radio-tracking study, we calculated that neighborhood size was 62.2 ha, which included between 204 and 480 individuals. Our population genetic analysis (based on randomly amplified polymorphic DNA (RAPD) and microsatellite markers) showed relatively low levels of genetic differentiation (Qpopulations[Formula: see text] 0.07–0.10) between subpopulations and no inbreeding within subpopulations (f = 0.0003). These estimates of population subdivision translate into an effective migration rate (Nem) of 2.3–3.3 per year, which represents a high level of gene flow. Invasion by exotics will reduce the overall productivity of the habitat, and will lead to isolation among subpopulations if favorable habitat patches become isolated.