Abstract <p>Large-scale habitat loss and degradation have triggered a global biodiversity crisis with ecological-specialist species at increased risk of extinction. The Rattlesnake-Master Borer (<i>Papaipema eryngii</i>) is a small noctuid moth whose larvae feed exclusively on rattlesnake master (<i>Eryngium yuccifolium</i>), a native grassland plant ranging throughout the eastern United States. Widespread loss of grassland habitats has resulted in declines of <i>P. eryngii</i> and the species has been considered for listing under the U.S. Endangered Species Act. We analyzed the mitochondrial <i>COI</i> barcode sequences of 76 <i>P. eryngii</i> from across the species’ distribution to examine genetic diversity and structure, and to test for historical changes in population size. We found 26 haplotypes with high haplotype diversity and low nucleotide diversity. Small but significant amounts of genetic differentiation occurred between populations separated by the Mississippi River and among representation units, but &gt; 90% of genetic variation occurred within populations. Isolation by distance was minimal, suggesting a lack of dispersal limitation, at least historically. All historical demographic analyses supported a recent population expansion, likely since the Last Glacial Maximum. Much genetic diversity is present within the range of <i>P. eryngii</i>, but the absence of some haplotypes from where they occurred historically suggests efforts to facilitate gene flow among extant populations may be needed to abate the loss of genetic diversity.</p> Implications for insect conservation <p>This study provides genetic data to inform management actions and future Species Status Assessments. Such data were unavailable for the previous Species Status Assessment, which based representation units on plant hardiness zones.</p>