Background: Tetranychus urticae is a highly polyphagous species with a cosmopolitan distribution that has the status of pest in more than 100 economically significant crops all over the world. Despite a number of previous effortsto isolate genetic markers, only a reduced set of microsatellite loci has been published. Taking advantage of the whole genome sequence of T. urticae that recently became available; we isolated and characterized a new set of microsatellite loci and tested the level of polymorphism across populations originating from a wide geographical area. Results: A total of 42microsatellite sequences widespread in the T. urticae genome were identified, the exact position in the genome recorded, and PCR amplification of microsatellite loci tested with primers defined here. Fourteen loci showed unambiguous genotype patterns and were further characterized. Three multiplex polymerase chain reaction sets were optimized in order to genotype a total of 24 polymorphic loci, including 10 previously published Tetranychus-specific loci. The microsatellite kits successfully amplified 686 individuals from 60 field populations for which we assessed the level of genetic diversity. The number of alleles per locus ranged from 3 to 16 and the expected heterozygosity values ranged from 0.12 to 0.81. Most of the loci displayed a significant excess of homozygous and did not model the Hardy–Weinberg equilibrium. This can be explained by the arrhenotokous mode of reproduction of T. urticae. Conclusions: These primers represent a valuable resource for robust studies on the genetic structure, dispersal and population biology of T. urticae, that can be used in managing this destructive agricultural pest.