Technical Abstract: We have developed an efficient method for generating new piggyBac insertions in the germline of F1 hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. We established seven independently-derived Minos-based piggyBac helper strains, each of which carries a single Minos element in which the Minos transposase gene is interrupted by a piggyBac transposase gene driven by the Drosophila hsp70 promoter. The donor strain is homozygous for a single piggyBac insertion element in which the transposase gene is interrupted by the 3xP3-EGFP reporter. In addition to the 3xP3-driven, eye-specific EFGP expression, the donor strain also shows muscle-specific EGFP expression due to the location of the piggyBac insertion in the 3'UTR of a muscle actin gene. Remobilization of the donor element and reinsertion at a new location is accompanied by loss of muscle-specific EGFP expression, but retention of eye-specific EGFP expression due to the presence of the 3xP3 promoter. All seven helpers tested in conjunction with this donor generated new insertion lines at a very high rate. In a pilot screen employing the M59 helper line, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating 411 new piggyBac insertion lines. Twenty-one (5%) of the 411 were homozygous lethal and several displayed enhancer-trap phenotypes. Excision footprints of donor chromosomes were sequenced for two independent excision events, and in both cases excision was precise, leaving no footprint. These results represent the first step in the development of an insertional mutagenesis system that will undoubtedly increase the power of transposon-based technologies for functional genomic studies in this coleopteran species.