Evidence for eustasy at the Kinderhookian-Osagean boundary is presented here in a new synthesis based on detailed analysis of the boundary in the central Appalachian Basin and regional mapping of the Kinderhookian-Osagean boundary unconformity in the conterminous United States. Detailed stratigraphic analysis within the central Appalachian Basin shows that coarse-grained, fluvial sandstones (Black Hand, Burgoon, Big Injun, Purslane) are associated with valley incision up to 60 m and a widespread sequence boundary (SB2) that is inferred to have resulted from a forced regression during the Kinderhookian-Osagean boundary interval. The extent of unconformity at the boundary was evaluated by mapping the inferred positions of shorelines during the Kinderhookian, Kinderhookian-Osagean boundary interval, and early Osagean based on stratigraphic data from Correlation of Stratigraphic Units of North America (COSUNA) charts. This analysis shows areas of extensive unconformity at the Kinderhookian-Osagean boundary across the United States inferred to be the result of sea-level fall and recovery during a period of ~2 m.y. or less, based on missing conodont zones. The Kinderhookian-Osagean boundary is equivalent in age to the global Tn2-Tn3 boundary, and supporting evidence for global regression and eustasy at this boundary is also reviewed. The combination of this evidence for eustasy with recently reported middle to early late Tournaisian (Tn2a-Tn3b) diamictites in South America permits the inference of continental glaciation at this time. Previous studies of oxygen and carbon isotope data from marine carbonates and fossils have shown strong positive anomalies suggestive of global cooling at the Kinderhookian-Osagean and Tn2-Tn3 boundary, providing further support for the hypothesis of continental glaciation in the late Tournaisian.