Aims: A new polymerase chain reaction (PCR) method for the identification of Bacillus sporothermodurans strains from sterilized or ultrahigh temperature-treated milk and milk products and from other non-milk sources and environments, including the dairy farm. Methods and Results: Two strains from raw milk and feed concentrate could be allocated to B. sporothermodurans based on 16S rDNA sequencing and DNA-DNA hybridization results. Two specific PCR primers were derived from the 16S rRNA gene of B. sporothermodurans . Conclusions: The PCR identification method was validated using a collection of B. sporothermodurans strains from different sources and on a large collection of dairy and non-dairy Bacillus spp. and other relevant taxa. Significance and Impact of the Study: This PCR method was used as a screening method for strains with very heat-resistant endospores, isolated at the dairy farm level after heat treatment for 30 min at 100degreesC. Seventeen strains isolated at the dairy farm were identified as B. sporothermodurans . They originated mainly from feed concentrate and also from soy, pulp and silage. The PCR identification method described here can, therefore, contribute to a better understanding of the route by which B. sporothermodurans contaminates raw and/or heat-treated milk.