The fault boundary between the western and eastern Blue Ridge (WBR-EBR) in the southern Appalachians separates Mesoproterozoic basement rocks and their cover from Neoproterozoic to Paleozoic accreted rocks. Several northeast striking faults delineate the boundary, including the Gossan Lead shear zone in northwestern North Carolina. Varying tectonic interpretations of WBR-EBR boundary include a premetamorphic fault, an Acadian dextral strike-slip fault, or an Alleghanian fault. We use field-based, microstructural, and theromochronometric analyses to determine the conditions, kinematics, and timing of deformation, in order to distinguish among competing hypotheses for the Gossan Lead shear zone. This comprehensive approach has allowed us to attribute a number of new and previously observed tectonic fabrics to specific orogenic events; key relationships necessary to the study of multiply deformed tectonic margins. Detailed mapping and microstructural analysis of the Gossan Lead shear zone document a several kilometer-wide mylonitic zone with kinematic indicators that record dominantly top-to-the-NW thrust motion, with local strike-slip and normal sense indicators. Dynamically recrystallized quartz and feldspar constrain a range of deformation conditions from amphibolite to greenschist facies. Two unaltered lineation-forming amphiboles from mylonitic amphibolites record ⁴⁰Ar/³⁹Ar cooling ages of 347–345 Ma, and a mylonitized metagraywacke records a muscovite ⁴⁰Ar/³⁹Ar cooling age of 336 Ma. These data are consistent with dominantly NW directed thrusting along the Gossan Lead shear zone at amphibolite to greenschist facies conditions, and rapid cooling in the Middle Mississippian. We suggest these data support overprinting and/or reactivation of an earlier structure along this complexly deformed boundary by 336 Ma.