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This research was undertaken to understand the replication kinetics of Salmonella enteritidis (S. enteritidis) in the internal organs of ducklings after oral challenge over a 2 wk period. A serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) assay was used to detect genomic DNA of S. enteritidis in the blood and the internal organs at different time points respectively. The results showed that the spleen was positive at 12 h post inoculation (PI) and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas and kidney were positive at 20 h PI, and the final organ to show a positive results was the gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h–36 h PI, with the liver and spleen containing the highest concentration of S. enteritidis. The blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 2 wk for the spleen without causing apparent symptoms. To make the results meaningful, a side-by-side bacteriology method (IFA) was performed. The results of IFA were similar to the FQ-PCR assay. This research provided a significant data for understanding the life cycle of S. enteritidis in the internal organs, and may help to understand the pathogenesis of S.entertidis in the future.