Principles of amino acid paleothermometry are used to estimate paleotemperatures and latitudinal temperature gradients for the period during and following the last glacial maximum in the Mississippi Valley. Gastropod shells were collected from the Peoria Loess for amino acid analysis, and Arrhenius parameters of isoleucine epimerization were determined experimentally for the gastropod genera Catinella and Hendersonia . There are 37 radiocarbon and 5 thermoluminescence ages that constrain the paleotemperature estimates and provide additional chronological data for loess deposition in the region. Amino acid paleotemperature estimates suggest that the north-south temperature gradient was significantly depressed in the Mississippi Valley for a considerable period during the past ≈25 k.y. Effective diagenetic temperature estimates indicate that at some time during or following the last glaciation, the effective-temperature gradient was ≈0.3–0.6 °C/degree of latitude, which is significantly lower than the modern mean annual air-temperature gradient of ≈0.9 °C/degree of latitude. Calculated effective paleotemperatures for three localities in Tennessee and Mississippi suggest that temperatures were ≈7–13 °C lower than present during the period from ca. 24 to 16 ka in the lower Mississippi Valley. These results provide additional evidence for a significant cooling in southern United States continental temperatures during the last glacial maximum.