Abstract: Abstract: 【Objective】The SNaPshot technique, a multiplex PCR method for genotyping SNP markers, is rapid, accurate and cost effective. In this paper, we used multiplex PCR and the SNaPshot technique to establish a multiplex primer assay for the SNaPshot reaction in Eucalyptus spp., in order to promote the application of SNPs in Eucalyptus spp. genetic linkage map construction and identification of the major commercial clones cultivated in China. 【Method】The primers were designed with Eucalyptus ESTs and the PCR products were sequenced using a PCR direct sequencing method to detect SNP loci. The selected SNP markers were used to establish the multiplex primer assay for the SNaPshot reaction. Finally, we applied this method to assay two parents and six F₁ mapping population sibs of E. urophylla × E. tereticornis, as well as 16 commercial Eucalyptus clones cultivated in China.【Result】Twelve SNP markers with three groups(I, II and III)were analyzed and the multiplex primer assay for the SNaPshot reaction in Eucalyptus was established. The SNaPshot assay genotyping results were in complete agreement with the PCR product direct sequencing results. The polymorphism diversity of these SNPs was also analyzed; all 12 loci were polymorphic among the six E. urophylla × E. tereticornis sibs and the commercial clones. The expected heterozygosity(H_e)ranged from 0.11 to 0.51, observed heterozygosity(H_o)ranged from 0.11 to 0.56, and polymorphic information content(PIC)ranged from 0.10 to 0.37, showing medium or low polymorphism.【Conclusion】The multiplex primer assay for the SNaPshot reaction established here is an efficient and accurate method for Eucalyptus genotyping, which will be useful for genetic linkage map construction and identification of Eucalyptus clones.