Colibacillosis is a disease in poultry caused by avian pathogenic Escherichia coli (APEC) strains which leads to great economic losses in the poultry industry. These E. coli strains contain various virulence genes which grant the bacteria the ability to proliferate in the poultry host and cause disease. Many genes which can contribute to virulence have been identified and can be used to screen E. coli strains to infer pathogenicity and aid in the identification and classification of APEC. Multiplex polymerase chain reaction methods were designed and optimized to rapidly detect 18 different virulence genes in E. coli strains that were isolated in South Africa and Zimbabwe from various sources, including from chickens showing signs of colibacillosis. Virulence gene profiles were constructed for each E. coli isolate from the multiplex data for the comparison of the colibacillosis isolates with the other isolates. The South African E. coli isolated from chickens with signs of colibacillosis showed higher virulence gene prevalence in comparison with the Zimbabwean and other samples except those isolated from chicken faeces. The multiplex polymerase chain reaction designed in the present study successfully screened E. coli isolates for various APEC-related virulence genes, including genes recently described in the literature.