Abstract: 【Objective】The distribution patterns and sequence characterstics of simple sequence repeat(SSR) in Eucalyptus cloeziana genome were analyzed in order to lay the foundation for developing SSR markers in molecular marker-assisted breeding of E. cloeziana.【Method】The genome sequence information of E. cloeziana was obtained based on high-throughput sequencing. Scaffold sequences were identified after rigorous filtration, splicing and assembly. The SSR loci from Scaffolds were searched and analyzed by using MicroSAtellite(MISA). Primer 5.0 software was used to design the SSR sequences with high potential for polymorphism development(the repetitions of dinucleotide ≥ 10, the repetitions of trinucleotides ≥ 7). Forty-eight genomic SSR primer pairs were randomly selected for validity detection and screening of primers successfully amplicated.【Result】The genome of E. cloeziana contained 177750 SSR loci, including 171530 single SSRs and 6220 compound-loci SSRs. The SSR occurrence frequency was 17.86% and the distribution density was 1/3.13 kb. There were abundant SSR motifs in E. cloeziana genome, and among them, the mononucleotide repeats were the most abundant types, accounting for 69.33% of the total genomic SSRs, followed by dinucleotide and trinucleotide repeats, accounting for 21.01% and 7.58%, respectively. The proportions of tetra -, penta-and hexa-nucleotide repeats were relatively low, and the total proportion of the three nucleotides was 2.08%. For the SSR motifs, AG/CT were the dominant primitive types(16812), followed by AT/AT(8193), AAG/CTT(4404). A total of 48 SSR marker pairs were randomly selected for initial screening test. Thirty-one primers pairs could effectively amplify clear and bright bands matching the expected size, and 14 of them showed polymorphism across six samples of E. cloeziana. The percentage of polymorphic loci was 29.17%.【Conclusion】The SSR loci in the genome of E. cloeziana occur in high frequency and abundant motifs, and exhibit great potential for developing polymorphic SSR markers. Therefore, the results provide a basis for further SSR development and screening, genetic diversity analysis and genetic mapping of E. cloeziana and other Eucalyptus trees.