Mycoplasma genitalium has been proposed as a suitable model for an in-depth understanding of the biology of a free-living organism. This paper reports that the expression of the aminoglycoside resistance gene aac(6′)-aph(2″), the only selectable marker hitherto available for M. genitalium genetic studies, correlates with a growth impairment of the resistant strains. In light of this finding, a tetM438 construction based on the tetracycline resistance gene tetM was developed; it can be used efficiently in M. genitalium and confers multiple advantages when compared to aac(6′)-aph(2″). The use of tetM438 significantly improves transformation efficiency and generates visible colonies faster. Finally, the improvements in the pMTn TetM438 construction made it possible to obtain insertions in genes which have not been previously considered to be dispensable under laboratory growth conditions. © 2006 SGM.