While real-time–polymerase chain reaction (RT PCR) has been used as a rapid test for detection of Salmonella Enteritidis in recent years, little research has been done to assess the feasibility of pooling poultry environmental samples with aSalmonella Enteritidis–specific RT PCR assay. Therefore the objective of this study was to compare RT PCR SalmonellaEnteritidis detection in individual and pooled (in groups of two, three, and four) poultry environmental drag swab samples to traditional cultural methods. The drag swabs were collected from poultry facilities previously confirmed positive forSalmonella Enteritidis and were cultured according to National Poultry Improvement Plan guidelines. Initial, SalmonellaEnteritidis–specific RT PCR assay threshold cycle cutoff values of ≤36, ≤30, and ≤28 were evaluated in comparison to culture. The average limit of detection of the RT PCR assay was 2.4 × 103 colony-forming units (CFUs)/ml, which corresponded to an average threshold cycle value of 36.6. Before enrichment, samples inoculated with concentrations from 102 to 105 CFUs/ml were detected by RT PCR, while after enrichment, samples inoculated from 100 to 105 CFUs/ml were detected by RT PCR. Threshold cycle cutoff values were used in the subsequent field trial from which Salmonella Enteritidis was cultured in 7 of 208 environmental samples (3.4%). Individual samples were 99.0%, 100%, and 100% in agreement with the RT PCR at threshold cycle (Ct) cutoff values of ≤36, ≤30, and ≤28 respectively. The agreement for pooled samples also followed the same trend with highest agreement at Ct ≤ 28 (pool of 2  =  100.0%, pool of 3  =  100.0%, pool of 4  =  100.0%), midrange agreement at Ct ≤ 30 (pool of 2  =  99.0%, pool of 3  =  100.0%, pool of 4  =  100.0%), and lowest agreement at Ct ≤ 36 (pool of 2  =  98.1%, pool of 3  =  97.1%, pool of 4  =  98.1%). In conclusion, regardless of the level of pooling after tetrathionate enrichment, sensitivity was very good, and results would be comparable to what would have been found with individual culture or individual RT PCR at Ct ≤ 36.