The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.