In this study, we have evaluated the performance characteristics (host-specificity and -sensitivity) of four human wastewater-associated Escherichia coli (E. coli) genetic markers (H8, H12, H14 and H24) in ten target (human) and non-target (cat, cattle, deer, dog, emu, goat, horse, kangaroo and possum) host groups in Southeast Queensland, Australia. The overall host-sensitivity values of the tested markers in human wastewater samples were 1.0. The overall host-specificity values of these markers to differentiate between human and animal host groups were 0.94, 0.85, 0.72, and 0.57 for H8, H12, H24 and H14, respectively. Based on the higher host-specificity values, H8 and H12 markers were chosen for a validation environmental study. The prevalence of the H8 and H12 markers was determined among human wastewater E. coli isolates collected from a wastewater treatment plant (WWTP). Among the 97 isolates tested, 44 (45%) and 14 (14%) were positive for the H8 and H12 markers, respectively. A total of 307 E. coli isolates were tested from environmental water samples collected in Brisbane, of which 7% and 20% were also positive for the H8 and H12 markers, respectively. Based on our results, we recommend that these markers could be useful when it is important to identify the source(s) of E. coli (whether they originated from human wastewater or not) in environmental waters.