Microsatellite markers and fingerprinting techniques have demonstrated great potential for assessing colony affiliation in Reticulitermes species. This study utilized microsatellite allele fingerprints and examined variation at eight microsatellite loci within five different R. flavipes collection sites in each of Lancaster and Madison counties in Nebraska. In the termite populations from Lancaster County, five of the eight microsatellite loci (Rf 1-3, Rf 3-1, Rf 5-10, Rf 6-1, and Rf 24-2) provided informative genotypic variability allowing colony distinction. DNA sequence data at locus Rf 24-2 also provided conclusive evidence that populations sampled were distinct colonies. Five microsatellite loci (Rf 3-1, Rf 5-10, Rf 6-1, Rf 11-1, and Rf 24-2) were also found to be informative in the termite populations from Madison County. Genotypic diversity by microsatellite allele assignment allowed distinctions between the most closely collected as well as distantly collected colonies.