Methods of gene stacking are described herein. The methods can be used to repeatedly add genes into a chosen locus in a precise manner, which ensures co-segregation of all introduced genes and contributes to the stabilization of gene expression. In addition, methods of removing any additional foreign DNA elements such as selectable markers are provided. Seed stocks or cell lines comprising a gene stacking site, vectors containing an insert flanked by target sites for a site-specific DNA recombinase for use in the methods and kits for carrying out the methods are also provided herein.