We used a balancer chromosome to recover ethylmethanesulfonate-induced recessive mutations in a targeted region of the genome of the red flour beetle (Tribolium castaneum) by the technique of chromosome extraction. The experiments reported herein constitute the first successful application of this powerful technique in the order Coleoptera. Using the balancer chromosome maxillopedia-Dachs3 (mxpDch-3), we recovered seven recessive visible variants representing seven distinct loci and several dozen recessive lethal variants representing at least five distinct loci after screening 1,607 EMS-mutagenized chromosomes. A subset of the mxpDch-3-extracted mutations were positioned on the map of the second linkage group by a series of two-, three-, and four-point crosses. The orientation of the homeotic gene complex (HOM-C) on this linkage group was also determined. With the advent of better and more varied balancer chromosomes and the concomitant improvement of chromosome extraction procedures for genetic analysis of T. castaneum, saturation mutagenesis of targeted regions of the genome is now feasible in this species.