The bacterial pathogen Paenibacillus larvae is the causative agent of American foulbrood disease in honeybees (Apis mellifera). A touchdown nested PCR protocol was developed to detect the presence of P. larvae spores directly in honey and hive samples. This approach allows early discovery of the bacteria even at concentrations below pathogenic levels, opening the door to new prophylactic approaches against American foulbrood and real-time epidemiological studies.