Leuctra Stephens, 1836 is the fourth most speciose genus of Plecoptera (Leuctridae) east of the Rocky Mountains with 31 recognised species, trailing only Isoperla Banks, 1906 (58), Allocapnia Claassen, 1928 (47), and Perlesta Banks, 1906 (34). Although Leuctra females are described in taxonomic literature, they are difficult to morphologically distinguish among regional congeners, and identifications are often made through inference only (i.e., presence of males). This is particularly problematic in the Appalachian Mountains of the United States of America, which host numerous Leuctra species. We sampled the stonefly fauna of Mount Mitchell, western North Carolina, United States of America, from April to October 2019. The mitochondrial cytochrome C oxidase subunit 1 gene was sequenced for 39 adult males and 239 adult females of Leuctra. This allowed us to confidently place species names on all of the latter individuals. Phylogenetic tree- and genetic distance–based methods consistently grouped females with males for nine recognised species. Two separate L. ferruginea (Walker, 1852) operational taxonomic units were recognised, albeit with low divergence values, and an additional undetermined Leuctra was identified based solely on females. Digital stereomicroscope images were taken from females of each species unit to identify variation among and between species. This approach allowed for a more robust assessment of regional biogeographic patterns.