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Abstract Aim To examine range-wide patterns of genetic diversity in association with range limits in a broadly distributed temperate lizard, and to identify the contributions of a series of environmental, geographical and historical variables to the observed patterns. Reduced genetic diversity may limit local adaptation in peripheral populations, thereby limiting their ability to adapt to marginal environmental conditions, possibly explaining the existence of temporally stable range limits. Location Various sampling locales throughout eastern and central USA and southern Ontario, Canada. Methods Genetic diversity of nuclear DNA microsatellites was estimated for each of 38 populations from across the range of eastern North America’s most broadly distributed lizard, the five-lined skink, Plestiodon fasciatus (Linnaeus, 1758). Results Local climatic conditions and an interaction between distance from range border and glaciation history best predicted a population’s present-day genetic diversity. Overall, peripheral populations had reduced genetic diversity relative to that of central populations, but this difference was attributable to the reduced genetic diversity in peripheral populations to the north and west that are not bordered by any obvious physical boundaries. Main conclusions Some, but not all, peripheral populations had reduced genetic diversity relative to that of more central populations, which probably arose through an interaction of ecological and historical factors. Peripheral populations that were bordered by an obvious boundary (e.g. an ocean) had higher diversity than peripheral populations that were not bordered by an obvious physical barrier to range expansion, suggesting that reduced intrapopulation genetic diversity is associated with range limits in the five-lined skink.