We used analyses of the strontium isotope (87Sr/86Sr) ratios of tooth enamel to reconstruct the migration patterns of fossil mammals collected along the Aucilla River in northern Florida. Specimens date to the late-glacial period and before the last glacial maximum (pre-LGM). Deer and tapir displayed low 87Sr/86Sr ratios that were similar to the ratios of Florida environments, which suggest that these taxa did not migrate long distance outside of the Florida region. Mastodons, mammoths, and equids all displayed a wide range of 87Sr/86Sr ratios. Some individuals in each taxon displayed low 87Sr/86Sr ratios that suggest they ranged locally, while other animals had high 87Sr/86Sr ratios that suggest they migrated long distances (> 150 km) outside of the Florida region. Mastodons were the only taxa from this region that provided enough well-dated specimens to compare changes in migration patterns over time. Pre-LGM mastodons displayed significantly lower 87Sr/86Sr ratios than late-glacial mastodons, which suggests that late-glacial mastodons from Florida migrated longer distances than their earlier counterparts. This change in movement patterns reflects temporal changes in regional vegetation patterns.