The USA is experiencing a prolific invasion of the wood-boring emerald ash borer, Agrilus planipennis. Native to Asia, this beetle completes its life cycle on ash trees and results in nearly complete mortality of all infested trees. In the present study, we examined the levels of genetic diversity and differentiation among eight populations of Fraxinus pennsylvanica (green ash) using five polymorphic microsatellite loci. Genetic information was used to design guidelines for the establishment of a seed collection sampling strategy to conserve the genetic diversity of ash trees. We found high levels of genetic diversity, as indicated by the allelic richness, both across the populations (16.4 ± 5.18 alleles per locus) and within them (8.03 ± 1.21 alleles per locus). The expected and observed heterozygosity was also high (0.805 ± 0.38 and 0.908 ± 0.04, respectively), and there was moderate genetic differentiation among the populations (F
ST= 0.083) with members of these eight populations grouped into three distinct clusters. We examined the relationship between the number of individuals sampled and the number of alleles captured in a random sample taken from a population of 10,000 individuals. Only sample sizes of 100 individuals captured most of the alleles (average = 78.74 alleles), but only seven of 50 samples effectively captured all the 82 alleles. Smaller samples did not capture all alleles. A probabilistic model was used to determine an optimal sampling strategy, and it was concluded that a collection of 200 seeds from each of five mother trees would have the highest likelihood of capturing all alleles in a population.