Influenza viruses from domestic aquatic birds can be transmitted to chickens, resulting in continued prevalence of the disease. H3 viruses are one of the most frequently identified subtypes in domestic ducks. Results from our previous serologic study suggested that H3 virus infections potentially exist in chickens with a wide geographical distribution in China. To better understand their pathogenic potential, two H3N8 influenza viruses isolated from domestic ducks were selected for experimental infections in chickens. We found that viral shedding lasted for at least 14 days postinfection for both viruses; however, one virus caused mortality in the chickens when coinfected with Escherichia coli. Sequencing of the viral HA gene isolated from the inoculated chickens revealed two amino acid mutations within the gene. These findings demonstrate the pathogenicity of the H3N8 domestic duck influenza viruses to chickens, highlighting the need for routine epidemiologic investigations of H3 subtype influenza viruses in chicken populations.