A Eucalyptus dunnii Maiden breeding population of 46 accessions originated in Australia and selected for fitness to subtropical and cold environments was screened by Amplified Fragment Length Polymorphism (AFLP) and microsatellite markers to obtain quantitative estimates of genetic diversity. A randomly chosen group of AFLP primers generated 205 AFLP bands that were used to fingerprint the genotypes and to evaluate genetic relationships among accessions. Sixty-eight percent (140) of the bands were polymorphic markers. The mean diversity index (DI) was 0.33 and about 52% of the loci had values greater than 0.4. Cluster analysis derived from similarity indices (SI) revealed no particular grouping among accessions suggesting the absence of closely related genotypes, except for five pairs of genotypes. Bootstrap analysis results confirmed the suitability of AFLP to describe genetic relationships in this breeding population. In addition, four highly informative microsatellites were used to construct an identification matrix that discriminated nearly all of the genotypes. Mean values for the number of alleles per locus, DI and SI among accessions were 13, 0.78 and 0.19, respectively, indicating that the breeding population has high genetic diversity. However, several genotypes showed the presence of single microsatellite bands suggesting a putatively important degree of homozygosity. Molecular data were used to design a clonal seed orchard. To achieve this aim, the nine most divergent pairs of genotypes were chosen, thereby retaining 95.2% of the total number of alleles from the 140 polymorphic AFLP loci and the four microsatellite loci analyzed. Mean DI and SI for AFLP and microsatellites showed no significant differences between the original breeding population and the selected seed orchard, confirming that a seed orchard can be designed with a limited number of individuals, which allows similar accessions to be discarded and avoids inbreeding.