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The elongate twig ant Pseudomyrmex gracilis is widely distributed in the New World ranging from southern USA to Brazil and northern Argentina. To investigate its within-colony relatedness, we developed primers for nine polymorphic microsatellite loci. High levels of within-population variation were observed, with number of alleles ranging from two to 13, and heterozygosity from 0.28 to 0.90. The loci showed no deviations from Hardy–Weinberg equilibrium and no linkage disequilibrium. [ABSTRACT FROM AUTHOR]