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Simple Summary: Tiger beetles (Coleoptera: Cicindelidae) are highly predatory and colorful insects of long-standing fascination by entomologists. Most species in this group are in decline from range-wide habitat loss, including those with wide ranges such as the cobblestone tiger beetle (Cicindelidia marginipennis (Dejean, 1831)). This species is considered threatened and/or endangered range-wide, and conservation activities are hindered by a lack of basic information about this species' biology. Here, we describe new aspects of this species' basic biology in the lab and the field and use the mitochondrial locus cytochrome oxidase I (COI) to examine biogeographic patterns. We present larval descriptions with detailed drawings and find genetic evidence for geographically structured populations, suggesting that further conservation genetic research is warranted for this species. We expect the new tools and information presented in this paper will allow specialists to test further hypotheses about this species, advance survey methods, and guide proactive tiger beetle conservation. The cobblestone tiger beetle, Cicindelidia marginipennis (Dejean, 1831) is a North American species specializing in riparian habitats from New Brunswick, Canada, to Alabama in the United States. In the United States, this species is state-listed as threatened or endangered range-wide and periodically receives consideration for federal listing, mostly due to habitat decline. Despite its conservation status, intraspecific genetic diversity for this species has not been explored and little is known about its natural history. To support further inquiry into the biology of C. marginipennis, this study provides the first look at range-wide genetic diversity using mitochondrial DNA (mtDNA), describes all three larval instars, and describes natural history characteristics from captive rearing and field observation. Based on mtDNA analyses, our results suggest that geographically based population structure may exist throughout the range, with individuals from Alabama possessing haplotypes not found elsewhere in our sampling. Further genetic analyses, particularly multi-locus analyses, are needed to determine whether the Alabama population represents a separate cryptic species. Our morphological analysis and descriptions of larval instars reveal a combination of characteristics that can be used to differentiate C. marginipennis from closely related and co-occurring species. Based on our field observations, we find that the larval "throw pile" of soil excavated from burrows is a key search image for locating larvae, and we provide descriptions and detailed photographs to aid surveys. Lastly, we find that this species can be successfully reared in captivity and provide guidelines to aid future recovery efforts. [ABSTRACT FROM AUTHOR]