Abstract: 【Objective】The aim of this study was to develop microsatellite （SSR） molecular markers for Lambdacyhalothrin resistant and susceptible strains of Neoseiulus barkeri， and to provide theoretical basis for genetic analysis of resistance genes and rapid identification and screening of resistant strains. 【Method】All unigenes from the transcriptome of N. barkeri were scanned using MISA software and its microsatellite （SSR） loci was screened by high-throughput. SSR primers were designed using Primer 5.0 software， 25 of these primer pairs were randomly selected for PCR amplification of the SSR loci genes for Lambda-cyhalothrin resistance and susceptible strains， and real-time fluorescence quantitative PCR gene expression analysis was conducted with 12 primer pairs with high reproducibility and stability. 【Result】A total of 52741 unigenes were obtained from the two strains， and the number and density of SSR loci were consistent. A total of 5483 SSR loci were detected from transcriptome， whose occurrence frequency was up to 8.11%， the analysis found that these loci were distributed in 4276 unigenes. SSR repeat types was mainly trinucleotide repeats， which accounted for 43.75% of the total number of SSR. A total of 81 kinds of repeat motifs were found， A/T was the most dominant motif in nucleotide repeats， accounting for 19.65% of the total， and 4570 SSR primers were designed， 23 pairs out of all 25 pairs randomly selected SSR primers could amplify the target gene fragments of both Lambda-cyhalothrin resistant and susceptible strains of N. barkeri. The results of real-time fluorescence quantitative PCR showed that the expression of 8 SSR loci genes of the Lambda-cyhalothrin resistant strains was higher than that of the susceptible strains ， and the relative expression of TRINITY_DN24111_c0_g1 gene was 4.11， extremely significantly higher than that of the susceptible strains （1.06）（P<0.001，the same below）. There were 4 SSR loci genes with lower relative expression than the susceptible strains， the relative expression of TRINITY_DN20624_c0_g1 gene was 0.68， extremely significant smaller than the susceptible strains（1.01）. 【Conclusion】The SSR loci in the transcriptome of N. barkeri are rich in information.The screened and developed 12 SSR specific primer pairs that express at different levels in N. barkeri resistant and sensitive lines can be applied to the rapid detection of Lambda-cyhalothrin resistance of N. barkeri.