To establish an in vivo transposition mutagenesis system of Saccharopolyspora,the transposition plasmids pJTn1pJTn6 were constructed by enhancing the expression of Tn5 transposase gene tnp (5) with strong promoters originated from Sa. spinosa and optimizing the Streptomyces Tn5based transposition plasmid pHL734. The results showed that plasmids pJTn1-pJTn6 transposed efficiently in Sa. erythraea. Plasmid pJTn1 and pJTn5 worked well in Sa.spinosa. Canonical events of Tn5 transposition were detected on 30 genomes out of 31 Sa.spinosa transposition mutants. There was a significant decrease in the yield of spinosad in eight mutants. It demonstrated that the pJTn plasmids are valuable tools for the transposition mutagenesis in the genus of Saccharopolyspora,which will facilitate studying the regulation of spinosad production and screening target genes for the high yield breeding.