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Genetic diversity in the nuclear genome of Eucalyptus nitens was assessed using 40 anonymous RFLP loci. Information about the RFLP probes was available from the genetic linkage map of the species and probes that detected single loci were selected to cover the whole of the genome. Several loci that were monomorphic in the mapping pedigree were also used to avoid bias towards polymorphic loci. Variation was determined for 20 individuals from each of eight populations covering the natural range of the species. The proportion of rare alleles detected in populations ranged from 21% to 49%. The level of diversity within the nuclear genome was high (AS = 6.9, PS = 92.5%, HT = 0.445) compared with other species where RFLP surveys have been conducted. The level of variation detected was higher for RFLP loci than for isozyme loci, but the patterns of genetic diversity were similar for both marker types. Population similarities based on RFLP analysis of the nuclear genome were not concordant with those based on the chloroplast genome.