BACKGROUND: The mechanism of pathogenesis and the role of virulence factors of avian pathogenic E. coli is still ill-defined. The ostrich industry is expanding, resulting in the interaction between poultry and ostrich. It is reported that the investigation of iss and bor virulence genes together, due to their structural and functional similarities, is valuable. Objectives: The investigation and comparison of presence of two genes involved in serum resistance, iss and bor, in E. coli isolated from apparently healthy ostriches and poultry with colibacillosis. Methods: As a cross-sectional study, E. coli was recovered from fecal samples of apparently healthy ostriches and poultry with colibacillosis, and iss and bor genes were screened and compared via PCR in E. coli isolates. Results: iss frequencies, with no statistical difference, were 50% and 64.4% in E. coli isolated from apparently healthy ostriches and poultry with colibacillosis, respectively (P>0.05). 31.8% and 15.6% of E. coli isolated from apparently healthy ostriches and poultry with colibacillosis were positive for bor, respectively, with no statistical difference (P>0.05). 11.1% of isolates from colibacillosis and 18.2% of isolates from apparently healthy ostriches feces, with no statistical difference, were positive for both genes (P>0.05). Conclusions: Equal statistical distribution of both genes, bor and iss, between apparently healthy ostriches and poultry with colibacillosis and the health level of studied ostriches indicated that E. coli isolated from ostrich, probably employs other virulence factors instead of bor and iss to establish a disease. This hypothesis needs to examine more virulence genes in ostrich-origin E. coli. In addition, the ostrich feces could be introduced as a source of iss and bor genes.