The objective of this study was to identify and understand the regular distribution pattern and primary penetration site for Salmonella Enteritidis (SE) in the gastrointestinal tract of ducks. An assay based on the serovar-specific DNA sequence of SE from GenBank, a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction, was developed for the detection of SE. We used this assay to detect genomic DNA of SE in the blood and gastrointestinal tract, including duodenum, jejunum, ileum, cecum, rectum, esophagus, stomach muscularis, and stomach glandularis, from ducks after oral challenge at different time points. The results showed that SE was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive 8 hr postinoculation (PI). The organism was detected in blood 12 hr PI, while the final organ to show a positive result was the stomach at 24 hr PI. The copy number of SE DNA in each tissue reached a peak at 24–36 hr PI, with the jejunum, ileum, and cecum containing high concentrations of SE, whereas the blood, duodenum, rectum, stomach, and esophagus had low concentrations. SE populations began to decrease and were not detectable at 2 days PI, but were still present up to 9 days PI in the jejunum, ileum, and cecum without causing apparent symptoms. By 3 days PI the cecum had significantly higher numbers of SE than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for SE. In conclusion, the results provided significant data for understanding the life cycle of SE in the gastrointestinal tract and showed that the jejunum, ileum, and cecum were the primary sites of invasion in normal ducks after oral challenge. This study will help to understand the mechanisms of action of SE infection in vivo.