As a theoretical basis for improvement of Eucalyptus urophylla in China, the genetic diversity and structure for advanced generation breeding populations were evaluated, and the changes of genetic diversity between improved generation and their parental generation were compared. The genotyping was performed on families sampled from advanced generation with 16 neutral microsatellite loci. The testing of Hardy-Weinberg equilibrium showed significant heterozygote deficits in two improved generation breeding populations. The expected heterozygosity of the 3 rd generation was very close to that of the 2 nd generation breeding population, indicating no heterozygosity had been lost, and the genetic diversity was consistent in two improved populations supported by Shannon's information index. Wright's fixed index of two advanced generations also indicated significant heterozygote in advanced generation breeding populations, compared with their parental generation. Most of the molecular variation was explained by the source of the families, both in two advanced generations, using analysis of molecular variances (AMOVA). The advanced generation breeding population of E. urophylla had a high level of genetic diversity after heavy artificial selection, consistently, and with no significant difference among generations.