Prevalence was estimated for Salmonella enterica serotype enteritidis (SE) in layer house environments (n = 200 layer houses) and house mice (n = 129 layer houses) in 15 states throughout the United States. Environmental swabs were collected from manure, egg belts, elevators, and walkways. Live-catch rodent traps were placed for 4–7 days. Swabs and house mice were submitted to the laboratory for bacterial culture. Overall, 7.1% of layer houses and 3.7% of mice were culture positive for SE. The highest prevalence was in the Great Lakes region of the United States, and no SE was recovered from houses or mice in the southeast region. Presence of SE in layer houses was associated with age/molting, floor reared pullets, and number of rodents trapped. Cleaning and disinfecting houses between flocks was associated with a reduced risk. The prevalence of SE in mice from environmentally positive houses was nearly four times that of mice from environmentally negative houses.