We investigated the potential for using long-archived wood samples extracted from archaeological contexts at four Mississippian Period (AD 900–1600) settlements in eastern Tennessee for tree-ring dating purposes. Sixteen wood samples recovered from prehistoric sites were analyzed to: (1) crossmatch samples from each site with the intent of determining the relative chronological order of sites, (2) establish a floating prehistoric tree-ring chronology for eastern Tennessee, (3) determine the applicability of dendrochronology in prehistoric archaeology in eastern Tennessee, and (4) establish a strategy for future research in the region. We succeeded in crossmatching only three of the 16 tree-ring sequences against each other, representing two sites relatively close to each other: Upper Hampton and Watts Bar Reservoir. The average interseries correlation of these three samples was 0.74 with an average mean sensitivity of 0.26, and they were used to create a 131-year-long floating chronology. The remaining samples contained too few rings (15 to 43) for conclusive crossmatching. Our results demonstrate that dendrochronological techniques may be applied to the practice of prehistoric archaeology in the Southeastern U.S., but highlight the challenges that face dendroarchaeologists: (1) poor wood preservation at prehistoric sites, (2) too few rings in many samples, (3) the lack of a reference chronology long enough for absolute dating, and (4) the lack of a standard on-site sampling protocol to ensure the fragile wood samples remain intact.