Reverse genetics is commonly used to identify and characterize genes involved in a variety of cellular processes. There is a limited set of positive selectable markers available for use in making gene deletions or other genetic manipulations in Cryptococcus neoformans. Here, we describe the adaptation of the Bacteriophage P1 Cre-loxP system for use in C. neoformans, and its application in the excision and reuse of the geneticin drug marker. This tool will allow investigators to make multiple, sequential gene deletions in the same strain, which should facilitate the analysis of multigene families.