Pyelonephritis-associated pili (Pap) are fimbrial adhesions that facilitate binding of UPEC to Gal(α 1-4)Galβ moieties contained in membrane glycolipids on human uroeipthelial cells and are associated with acute kidney infection (pyelonephritis). In this study, pap phase transition frequencies were measured in clinical isolates for the first time and were shown to be markedly higher than the frequencies displayed by the same pap operons measured in E. coli K-12. In this relevant regulatory context, phase variation frequencies of homologous pap operons were found to be differentially affected by culture conditions, indicating a hierarchy of expression depending on environmental signals. Cross-talk between pap operons was also found to be dependent on culture conditions. The molecular mechanism of different phase variation frequencies between homologous pap operons was investigated by sequencing 82 pap regulatory regions and their regulators (papI and papB) from 54 UPEC isolates of different clinical origin (asymptomatic vs. symptomatic UTI).  The most variable region identified was a high affinity binding site for the pap autoregulatory protein PapB. The site contained a variable number of 9 bp repeats with (T/A)₃ sequences, which affected PapB binding and the frequency of off-to-on phase transition., under particular environmental conditions. Sequence diversification via point mutation was also observed among papI genes, encoding for the pap transitional activator PapI, and were shown to be under positive selection (Dn/Ds > 1) for functionally adaptive amino acid replacements. Certain PapI variants correlated with symptomatic disease and differed in their ability to activate pap operons. The ability of UPEC to co-ordinate expression of multiple surface factors is critical for the successful colonisation of the many complex micro-environments encountered in the human urinary tract.