To study the prevalence and isoforms of the pathogenicity island ETT2 among pathogenic Escherichia coli, as well as to determine the relationship between the ETT2 locus and other virulence factors, PCR amplifications target to the 35 ETT2-associated genes were established and used to investigate the presence of the ETT2 locus in 168 E. coli isolates from weaned piglets with edema and/or diarrhea or dairy cows with mastitis. The results showed that the ETT2 locus could be identified in the pathogenic E. coli isolates from colibacillosis in pigs and in the ones from mastitis in cows, but the presence of ETT2 among the isolates of porcine origin were significantly higher (85.87%) than that (47.37%) of bovine origin. Furthermore, 11 ETT2 isoforms were found in this research, including an intact form and 10 deletion types. The intact ETT2 was the prevalent form among the pathogenic E. coli isolates of porcine origin, and highly associated with the presence of shigatoxin type 2e (Stx2e), while the great majority isolates of bovine origin just carried various deletion types, and no distinct association with other virulence factors, e.g., the presence/absence of LT1, ST2, Cnf2, Tra, HPI, Hly, and F17a fimbriae.