Aims:The aim of this was to isolate Escherichia coli from broilers infected with chronic respiratory disease (CRD) and investigate the prevalence of outer membrane protein T (ompT) and increased serum survival (iss) genes. Methodology and results: Fifty trachea and lung samples were cultured for isolation of E. coli and molecular confirmation was done for these isolates using conventional PCR. Multiplex PCR technique was used to detect the existence of ompT and iss genes of E. coli. Results showed that the percentage of E. coli isolation reached 42% from all trachea and lung samples by using conventional procedures. However, the polymerase chain reaction (PCR) diagnosis method recorded 28% when using the uidA gene (genus-specific gene) for E. coli. Neither virulence genes (ompT and iss) were detected in all E. coli isolated in this study. Conclusion, significance and impact of study: Escherichia coli is the principal causative agent of CRD infections, which can bear different combinations of virulence genes that play significant roles in the pathogenicity of bacteria. All isolates in the existing study underwent APEC strains that didn't contain virulence genes like ompT and iss.