An existing microsatellite genotype database has been used for several years in population genetic assignment analyses of boll weevils, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), captured in eradication zones. It is important to update it in case of changes in genotype frequency at any of the locations over time. Such changes at neutral loci could be caused by drift, immigration, or population bottlenecks. We examined allele frequency distribution for 10 microsatellite loci to determine genetic differentiation among 10 boll weevil populations sampled from Texas and Mexico in 2009. In addition, temporal changes in genetic composition were examined in the eight populations for which samples were available from previous years. Substantial levels of spatial genetic structure were observed, with the 10 populations clustering as four major groups. Pairwise FST estimates in 2009 samples ranged from 0.001 (College Station-Cameron) to 0.492 (College Station-Ojinaga). There was little change in genetic profiles over time at four of the eight locations. Thus, for those four locations, genotype and allele frequency data can be pooled over the two sample dates, which will provide greater statistical power in future population assignment tests. However, genetic profiles changed substantially at Ojinaga, and to a lesser extent at Uvalde, Cameron, and Rosales, so the 2009 genotype data should be substituted in future analyses. Finally, populations from two new locations, Brownsville and Lockhart, TX, were sampled, genotyped, and added to the database. The addition of Lockhart is particularly important given its surprisingly high differentiation from the relatively nearby populations of Cameron and Uvalde.