Here, we document a technique to reduce the size of the genome of Pseudomonas putida by using a combinatorial mini-Tn5-targeted Flp-FRT recombination system. This method combines random insertions with the site-specific Flp-FRT recombination system to generate successive random deletions in a single strain in which parts of the genome are excised via the action of the cognate flippase. For this purpose, we have generated two mini-Tn5 transposon mutant libraries with single and double integrations of either mini-Tn5 KpF alone or mini-Tn5 KpF in parallel with mini-Tn5 TF, respectively. These mini-Tn5 transposons carry different selectable markers and each has an FRT (Flippase Recognition Target) site. Mapping of the position of both mini-Tn5 transposons in the chromosome of P. putida was conducted by Arbitrary Primed-PCR (AP-PCR). Subsequent sequencing of the PCR fragments led to the identification of the coordinates of the transposons and the orientation of both FRT sites. Under specific laboratory conditions, both FRT sites were recognized by the flippase, and the deletion of a nonessential intervening genomic segment along with the transposon backbones occurred without inheritance of any marker genes.