We present an optimized multiplex of 14 microsatellite loci that can be amplified in four reactions and loaded as two separate panels on the ABI genetic analyzer. The assay was validated on 342 individuals of field cricket (Gryllus campestris, L., 1758) from natural populations. The chosen loci are polymorphic and allow for efficient population genetic analysis, although we observed some aberrations, which are discussed. The optimized multiplexes sets show reproducible amplification pattern with low genotyping error rate and should be therefore very useful in analyzes of both experimental as well as natural populations of crickets. The optimized multiplex outperforms previously developed sets as it allows for amplification of 14 loci in four instead of seven reactions. These panels may serve as an applicable tool in conservation programs in countries in which this species is endangered.