The increased incidence of tuberculosis (TB) gave impetus for the increased interest in the study of mycobacterial genetics, which culminated in the publication of the full genome sequence of many mycobacterial strains. Since then, many genes and open reading frames of unknown function have been described and the expression of their encoded proteins is critical toward understanding the pathogenesis of TB and developing therapeutic and preventive strategies. Therefore there is an increased need for highly efficient methods for cloning of mycobacterial genes, as the limited cloning flexibility of current Escherichia coli-mycobacteria shuttle vectors remains a frequent impediment in genetic manipulation of mycobacteria. In order to overcome this limitation, we have converted representative extrachromosomal and integrative vectors into multiple destination mycobacterial vectors for one-step and restriction enzyme-free recombination cloning methodology that uses in vitro site-specific recombination. We provide several examples that highlight the potential of recombination cloning for gene expression in slow and fast-growing mycobacteria. Thus, a gene of interest can be transferred by simple recombination into our mycobacterial destination vectors, which serve a multitude of functional genomic studies.