Four microsatellite loci have been characterised in Eucalyptus nitens Maiden and in six other eucalypt species. The dinucleotide repeats were identified by screening a Sau3AI genomic DNA library from E. nitens with (CA)n and (GA)n oligonucleotide probes and sequencing the positive clones. Genetic analysis of 20 unrelated individuals from five populations of E. nitens showed all loci to be highly polymorphic with an average of 9.5 alleles per locus and an average heterozygosity of 0.575. Analysis of four individuals from each of six species from three subgenera showed complete conservation of microsatellite loci between species within the same subgenus, Symphyomyrtus, and conservation of 50% of loci across species between the two main subgenera, Symphyomyrtus and Monocalyptus. None of the primers amplified microsatellite loci in Eucalyptus maculata from the subgenus Corymbia. All microsatellite loci that were detected were polymorphic. Highly polymorphic microsatellite loci that are conserved across species will be useful for mapping quantitative traits, fingerprinting breeding lines, and for within-population studies requiring fine-scale analysis of genetic variation.