Concern by consumers about food safety has resulted in increased pressure on poultry companies to develop effective sanitation programs. Salmonella isolates in hatcheries are often the same species isolated from processing plants. Resistance develops in bacteria after prolonged exposure to disinfectants. The methods available in published literature to detect the efficacy of disinfectants are labor intensive and do not consider how bacteria behave when adhered to a solid surface. We used a recently developed technique, which utilizes the actual surfaces on which the disinfectant is to be applied, to evaluate the degree of resistance to four commercially available disinfectants of 17 bacterial isolates from poultry hatcheries. We found that bacterial isolates within the same genus and species have different sensitivities to the same disinfectant. In addition, disinfectants with similar but not identical chemical formulations have different efficacies against the same bacteria.