The western flower thrips, Frankliniella occidentalis (Pergande), is an invasive species and the most economically important pest within the insect order Thysanoptera. For a better understanding of the genetic makeup and migration patterns of F. occidentalis throughout the world, we characterized 18 novel polymorphic EST-derived microsatellites. The mutational mechanism of these EST-SSRs was also investigated to facilitate the selection of appropriate combinations of markers for population genetic studies. Genetic diversity of these novel markers was assessed in 96 individuals from three populations in China (Harbin, Dali, and Guiyang). The results showed that all these 18 loci were highly polymorphic; the number of alleles ranged from 2 to 15, with an average of 5.50 alleles per locus. The observed (H-O) and expected (H-E) heterozygosities ranged from 0.072 to 0.707 and 0.089 to 0.851, respectively. Furthermore, only two locus/population combinations (WFT144 in Dali and WFT50 in Guiyang) significantly deviated from Hardy- Weinberg equilibrium (HWE). Pairwise FST analysis showed a low but significant differentiation (0.026 < F-ST < 0.032) among all three pairwise population comparisons. Sequence analysis of alleles per locus revealed a complex mutational pattern of these EST- SSRs. Thus, these EST- SSRs are useful markers but greater attention should be paid to the mutational characteristics of these microsatellites when they are used in population genetic studies.