Sprouts have been implicated in several outbreaks of salmonellosis in the United States, where the seeds are thought to be the primary source of contamination. The objectives of this study were to investigate the influence of growth temperatures of Salmonella and storage temperatures of alfalfa seeds on heat inactivation of the pathogen on seeds. Alfalfa seeds were inoculated with a composite of Salmonella, grown at temperatures of 15, 25, 35 or 40C, stored at 22C for 7 days, dry heated at 65C for up to 48 h, chilled and subsequently microbiologically analyzed. The heat resistance of Salmonella at 65C (D65) varied with the growth temperatures in the order of 15C > 25C > 35C > 40C. The effect of storage temperature of seeds on heat inactivation of Salmonella was subsequently investigated. Inoculated seeds were dried, stored for 7 days at different temperatures (22, 15, 10, 4, 0, −10 or −18C) and then heat-treated at 60, 65 or 70C. Heat inactivation of Salmonella was dependent on the storage temperature and increased in the order of 0C ≈ −18C > 4C > 10C ≈ −10C > 15C > 22C. Storage at −18 or 0C followed by dry heating at 60 for 72 h consistently inactivated the pathogen to below detectable levels (<0.7 log cfu/g) and did not significantly (P > 0.05) reduce the sprouting yield of seeds compared to their untreated counterparts. These observations will be useful when developing effective strategies and practices to enhance the microbiological safety of alfalfa sprouts. Practical Applications Sprouts are very young shoots of plants often consumed raw in salads or sandwiches. Common sprouts include mung bean, soybean and alfalfa sprouts. Unfortunately, alfalfa sprouts have been implicated in outbreaks of gastrointestinal illness where the seeds themselves are contaminated with important disease-causing microorganisms. In this study, the influence of growth temperature of Salmonella and the storage temperature of seeds on the susceptibility of the disease-causing microorganism to heat was investigated. Storage of seeds in the refrigerator or freezer for a week prior to heating for 3 days completely destroyed the microorganisms and did not have any considerable impact on the seed viability. These observations will be useful for the sprouting industry to design and optimize heat processes to improve the safety of alfalfa sprouts.