HorologionValentine, one of the rarest and most enigmatic carabid beetle genera in the world, was until now known only from the holotype ofHorologion speokoitesValentine, discovered in 1931 in a small cave in West Virginia. A single specimen of a new species from Virginia was collected in 1991, but overlooked until 2018. DNA sequence data from specimens of this new species,Horologion hubbardisp. nov., collected in 2022 and 2023, as well as a critical examination of the external morphology of both species, allow us to confidently placeHorologionin the supertribe Trechitae, within a clade containing Bembidarenini and Trechini. A more specific placement as sister to the Gondwanan Bembidarenini is supported by DNA sequence data. Previous hypotheses placingHorologionin or near the tribes Anillini, Tachyini, Trechini, Patrobini, and Psydrini are rejected. The existence of two species ofHorologionon opposite sides of the high mountains of the middle Appalachians suggests that these mountains are where the ancestralHorologionpopulations dispersed from, and predicts the discovery of additional populations and species. All specimens ofH. hubbardiwere collected in or near drip pools, and most were found dead, suggesting that the terrestrial epikarst, rather than caves, is the true habitat ofHorologion, which explains their extreme rarity since epikarst has not been directly sampled. We recognize the tribe Horologionini, a relict lineage without any close relatives known in the Northern Hemisphere, and an important part of Appalachian biodiversity.