Site-specific recombination mediates the rearrangement of nucleic acids by the virtue of an recombinase acting on specific recognition sequences. Recombining activities belong either to the tyrosine- or serine-type group, based on the presence of specific residues in the catalytic centre, which can be further subdivided into families due to additional criteria. The most prominent systems are the λ phage integrase acting on att sites; the Cre recombinase from bacteriophage P1 with its loxP attachment sites; the FLP/FTR system of fungal origin, where it is required for 2-μm plasmid replication/amplification in yeast; and the prokaryotic β-recombinase that recombines six sites specifically in cis. Each of these has been exploited in fungal hosts of biotechnological, medical or general relevance, mainly for cloning projects, approaches of gene targeting, genome modification or screening purposes. With their precise and defined mode of action are site-specific recombination systems eminently suited for genetic tasks in fungi, like they are executed in functional studies at high throughput or modern approaches of synthetic biology.