Cryptococcus neoformans causes fatal meningoencephalitis if not timely treated. Targeted gene disruption for functional analysis of a gene involves overlap PCR for the production of gene disruption cassettes carrying dominant selectable markers, followed by biolistic transformation. However, the conventional overlap PCR method between two flanking regions of the target gene and selectable marker is often inefficient due to the long length of the PCR product and the presence of multiple templates. Here we describe double-joint PCR with split dominant selectable markers for the more convenient generation of a gene-disruption cassette in C. neoformans with high targeted integration frequency (Kim et al., Biochem. Biophys. Res. Commun 390(3):983-988, 2009).