Leghorn hens were infected with Salmonella Enteritidis cultures of known genomic content and subpopulation characteristics to determine comparative abilities to colonize the avian reproductive tract. Group 1 received phage-type (PT)4 22079, which is a dimorphic subpopulation that can both contaminate eggs and form biofilm. Group 2 received a 90:10 mixture of monomorphic PT13a strains 21027 and 21046, which produce biofilm or contaminate eggs, respectively. Group 3 received a 10:90 mixture of the same two PT13a strains, respectively. Trials were repeated three times and a total of 30 hens per treatment group were infected. Dosage was by oral gavage and was calculated as 8.6 ± 2.01 × 10⁷ colony-forming units per hen. Liver, spleen, and three different sections of oviduct (ovary, upper oviduct, and lower oviduct) were cultured per bird. Results were that all three groups had livers and spleens that were mostly positive (90.0% and 94.4% of 270 hens cultured, respectively). Reproductive-tract organs yielded 75 positives from 270 hens (27.8%), and treatment groups ranged from a low of 6.7% to a high of 76.7% positive cultures in any one trial. There was no significant difference between the numbers of positive reproductive-tract samples between treatment groups due to variance. These results suggest that the status of the reproductive tract at the time of infection may impact recovery of culture-positive tissue and contribute to variance. It is suggested that Salmonella Enteritidis cultures that vary in subpopulation composition have subtle differences in colonization of reproductive tissue that contribute to variance in egg contamination. Culture of non–reproductive-tract organs such as the liver and spleen was overall more reliable for detection of infected hens. The spleen was especially useful for detection because of its small size. Further research is needed to determine how sex hormones influence the infection pathway that results in egg contamination.