Standard population genetic analyses are implemented for a series of precontact and contact period samples from central and northern Florida to investigate changes in genetic variability and population affinity coincident with the establishment of Spanish missions during the 17th century. Estimates of FST based on odontometric data indicate limited heterogeneity for the Apalachee samples, suggestive of some degree of within-group endogamy for this ethnic group prior to contact. This corresponds well with ethnohistoric reconstructions indicating that Apalachee were populous, partially linguistically isolated from its neighbors, and involved in persistent cycles of warfare with neighboring groups. Estimates of extralocal gene flow for the Apalachee samples indicate limited initial changes in the mating structure of these populations. After 1650, however, extralocal gene flow increases, consistent with evidence for dramatic population movements throughout northern Florida and increased Spanish presence in the province, particularly at the mission of San Luis. Inclusion of non-Apalachee outgroups does not increase estimates of genetic heterogeneity, as was expected based on ethnohistoric data. The pattern of genetic distances suggests a biological division between north and south Florida population groups, consistent with archaeological and ethnohistoric data, and similarly indicates some distinction between precontact and postcontact local groups. Differential extralocal gene flow experienced by pre-1650 Apalachee and Timucua populations suggests localized mission experience. The Apalachee, with large, dense populations, experienced limited initial changes in genetic diversity or mating structure. However, after 1650 they were apparently involved in a much more expansive mating network that may have included Spaniards and immigrant Native American groups to the region. These results are in contrast to the mission experience of the Guale Indians of the Georgia coast. Am J Phys Anthropol, 2005. © 2005 Wiley-Liss, Inc.