Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance. Staphylococcus aureus has been extensively studied as a model pathogen. A plethora of methods and molecular tools has been developed for genetic modification of at least ten different staphylococcal species to date. Here we review recent developments of various genetic tools and molecular methods for staphylococcal research, which include reporter systems and vectors for controllable gene expression, gene inactivation, gene essentiality testing, chromosomal integration and transposon delivery. It is furthermore illustrated how mutant strain construction by homologous or site-specific recombination benefits from sophisticated counterselection methods. The underlying genetic components have been shown to operate in wild-type staphylococci or modified chassis strains. Finally, possible future developments in the field of applied Staphylococcus genetics are highlighted.