Abstract: Orthogonal design was applied to optimize SCoT-PCR amplification system of Citrus in five factors such as Mg2+，dNTPs，primer，Taq DNA polymerase and template DNA. An optimal reaction system of Citrus was established，PCR reaction mixtures contained Mg2+ 1.5 mmol · L-1，dNTPs 0.35 mmol · L-1，primers 0.625 μmol · L-1，Taq polymerase 0.25 U，template DNA 10 ng. The most suitable annealing temperature of primers was 49 ℃. Amplifications and genetic analysis were carried out on four tetraploids of Shatianyou pomelo [Citrus grandis（L.）Osb.‘Shatianyou’]and progenies from Wan2 [tangor，Citrus unshiu（Mark.）Marc. × Citrus sinensis（L.）Osb.] × Li2[sweet orange，Citrus sinensis（L.）Osb.]. The size range was typically between 150–2 100 bp. The 7 primers generated a total of 74 fragments from Wan2，Li2 and their progenies，48（64.9%）were polymorphic，and 18 progenies could be grouped into five distinct families based on similarity of 0.85，which indicated that greater genetic diversity was existence in the hybrids. And 84 bands were amplified from Shatianyou pomelo with 11 primers，of which 46（54.8%）were polymorphic. UPGMA cluster analysis showed that the similarity coefficient of five accessions ranged from 0.785 to 0.880. Four tetraploids could be grouped into three distinct families based on similarity of 0.825，and three homologous tetraploids were clustered in different locations，which showed that different degrees of genome variation was detected in tetraploids compared with the diploid. The SCoT system established in this report is useful for genetic diversity analysis of Citrus，which possesses considerable potential for citrus breeding. 　　

Key words: Citrus, start codon targeted polymorphism, genetic analysis, polyploid