Fluorescent proteins, especially green fluorescent protein (GFP), have been instrumental in understanding urinary tract infection pathogenesis by uropathogenic Escherichia coli (UPEC). We have used a recently developed GFP variant, vsfGFP-9, to create new plasmid- and chromosome-based GFP derivatives of the UPEC strain UTI89. The vsfGFP-9 strains are nearly 10× brighter with no in vitro growth or in vivo virulence defects compared to previously reported GFP-expressing UTI89 strains. The chromosomal vsfGFP-9 strain is equivalent to the wild type UTI89 during in vivo UTI, while both plasmid GFP constructs have an equivalent virulence defect compared to non-plasmid carrying UTI89. These new vsfGFP-9 expressing strains should be useful for further studies of the pathogenesis of UTI89, and similar strategies can be used to create improved fluorescent derivatives of other UPEC strains.