Gene pyramiding has been successfully practiced in plant breeding for developing new breeds or lines in which favorable genes from several different lines were integrated. But it has not been used in animal breeding, and some theoretical investigation and simulation analysis with respect to its strategies, feasibility and efficiency are needed before it can be implemented in animals. In this study, we used four different pure lines of Drosophila melanogaster, each of which is homozygous at a specific mutant gene with a visible effect on phenotype, to simulate the gene pyramiding process and analyze the duration and population size required in different pyramiding strategies. We finally got the ideal individuals, which are homozygous at the four target genes simultaneously. This study demonstrates that gene pyramiding is feasible in animal breeding and the interaction between genes may affect the final results.