Nine polymorphic microsatellite loci were isolated and characterized in the ant Crematogaster scutellaris from Italy. Allelic variation was tested for individuals collected from 26 nests in an olive grove in Tuscany, where ant–homopteran mutualistic interactions are under study to control damages to the olive crop. Allelic diversity was 12.4 and the number of alleles per locus ranged from 8 to 17. Mean observed and expected heterozygosity were 0.826 (range, 0.760–0.923) and 0.829 (range, 0.736–0.869), respectively. This novel set of loci will be used to assess population structure, dispersal strategies and mating system of C. scutellaris, and in particular to determine genetic relationships among individuals engaged in intraspecific conflicts.