Autofluorescent proteins (AFPs), such as green fluorescent protein (GFP) and DsRed, are valuable tools for studying plant-microbe interactions. Nevertheless, because of some limitations, efforts are ongoing to generate improved AFP variants. Several groups have generated variants of GFP with altered spectral characteristics, and faster maturing and brighter variants of DsRed. In this study we used plasmid and chromosomal constructs to test the efficacy of a new variant of DsRed, DsRed.T3_S4T, in Pseudomonas fluorescens F113rif. In addition, we compared the ecological fitness of strains carrying chromosomal copies of EGFP, DsRed or DsRed.T3_S4T. Strains expressing DsRed.T3_S4T fluoresced significantly brighter than strains expressing DsRed. Furthermore, it was found that although all strains grew equally well in vitro, only strains carrying DsRed.T3_S4T functioned as well as wild type in a competitive rhizosphere colonization assay. In particular, it was observed that DsRed.T3_S4T is an improved marker over DsRed for microbial ecology studies in this strain.