Combining a biotin-enrichment protocol and 454GS-FLX titanium pyrosequencing technology, we characterised 22 polymorphic microsatellite loci from the parasitic wasp, Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a cosmopolitan species commonly used in biological control against a wide range of both major lepidopterous pests of stored products and field crops in different parts of the world. Three multiplex PCR sets were optimised and characterised across 46 H. hebetor specimens from two samples collected from millet fields in Niger. Two to 11 alleles were found per locus and observed heterozygosity ranged from 0.289 to 0.826. Polymorphism was detected in both samples with a similar level of observed heterozygosity (0.482 vs. 0.502) and number of alleles (4.1 vs. 3.6). Deviation from Hardy-Weinberg equilibrium was detected at the same five loci in both samples and five or seven more loci in each sample but was not associated with heterozygote deficiencies. Even though evidence for linkage disequilibrium was found between a few alleles, these new loci segregated independently. The variability of the 22 loci will enable estimates of genetic diversity and structure patterns, as well as gene flow between H. hebetor populations at different spatial scales. Cross-species amplifications were successful among the six Bracon spp. tested and nine loci will be particularly appropriate for population genetic studies in B. brevicornis.