The analysis of craniometric data collected from skeletal remains, combined with archaeological data, can provide very valuable information pertaining to biological distance and gene flow among prehistoric populations through space and time. The goal of this study was to examine microevolutionary change among prehistoric populations in south Florida based on the degree of cranial variation among populations at seven prehistoric sites. It was expected that as time progressed, microevolutionary forces caused significant changes in the crania of the various populations based on the relative geographic proximity of the sites and the temporal distance between sites. A Microscribe 3-DX digitizer was used to collect coordinate data using the full protocol of cranial landmarks. Twenty-three interlandmark distances for n=223 skulls from seven sites, ranging in age from 8120 B.P. to 260 B.P., were analyzed using Cluster Analysis, an Analysis of Variance (ANOVA), a Tukey’s Pairwise Comparison (post-hoc test), a Multiple Analysis of Variance (MANOVA), and Principal Components Analysis (PCA) in SPSS 18.0. The seven sites were Windover (8Br246), Perico Island (8Ma6), Captiva Island (8Ll57), Belle Glade (8Pb40), Horr’s Island (8Cr41), Safety Harbor (8Pi2), and Fuller Mound A (8Br90). Of the 223 crania used, zero (0) skulls were 100% complete. Results of univariate and multivariate statistical analyses indicate that there are significant differences among the seven groups. The agglomerative cluster analysis did not provide significant results. When using Maximum Cranial Length (GOL), the ANOVA (F=5.190, p ≤ 0.000) and post-hoc tests indicated that there was a significant amount of variation among the seven populations. In a series of 12 MANOVA tests, it was determined that significant variation existed between Windover and each of the remaining six sites (F > 5, p ≤ 0.000). Additionally, the MANOVA tests indicated that significant variation existed between Horr’s Island and Safety Harbor (F = 8.151, p ≤ 0.000) and between Safety Harbor and Fuller Mound A (F = 5.549, p ≤ 0.000). Last, a Principal Components Analysis demonstrated that measurements consistent with length or breadth accounted for the largest percentage of variation among the populations. In conclusion, the data strongly demonstrate a significant amount of variation among prehistoric populations as time progressed from 8120 B.P. to 260 B.P. Specifically, changes in gene flow which can be attributed to significant differences among populations based on craniometric data parallel major time gaps and historical events in Florida. More generally, these results can be applied to other past populations to investigate similar patterns of gene flow and changes that may have occurred due to various social, political, and environmental stressors.