ABSTRACTFluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such asStenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7delivery plasmids for labeling ofS. maltophiliawith sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7transposons into single neutral sites located on average 25 nucleotides downstream of the 3′-end of the conservedglmSgene of differentS. maltophiliawild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence inGalleria mellonella. It is also shown that the mini-Tn7elements remained stably integrated in the genome ofS. maltophiliaover a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7delivery plasmids are valuable tools for generating fluorescently labeledS. maltophiliastrains that are indistinguishable in their properties from their parental wild-type strains.IMPORTANCEThe bacteriumS. maltophiliais an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence ofS. maltophiliainfections worldwide. One important virulence-related trait ofS. maltophiliais the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system forS. maltophiliato study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.