Data regarding the hemagglutination (HA) patterns of the three variants (classes I, II, and III) of the Escherichia coli adhesin PapG are conflicting. These HA patterns usually have been assessed for each papG allele separately with recombinant strains in slide HA assays. We rigorously evaluated an alternative microtiter tray HA assay and then used it to assess the HA of four erythrocyte types (human A1P1 and OP1, rabbit, and sheep erythrocytes) by multiple wild-type E. coli strains representing the four naturally occurring combinations of the papG alleles, i.e., class I plus III, class III only, Class II plus III and class II only. The microtiter tray HA assay displayed significantly better reproducibility of intraobserver (83%) and interobserver (86%) results than did slide HA assays (39 and 73%, respectively). Novel findings from the study of 32 wild-type P-fimbriated strains included reproducible determinations of phenotypic diversity among different papG categories, among strains within each papG category, and from day to day for individual strains. There was also substantial overlap of phenotypes between papG categories I plus III and III only and between II plus III and II only. A class III papG recombinant strain's HA pattern differed significantly from that of the wild-type class III strains. These data demonstrate that HA phenotypes of wild-type P-fimbriated E. coli strains can be reproducibly assessed by a microtiter HA assay and that they correspond broadly to papG genotype but in a more complex and varied fashion than previously recognized.