Apis mellifera jemenitica, the only indigenous honey bee race of Saudi Arabia, is well adapted to the harsh local environmental conditions. A large-scale field survey was conducted to screen major Saudi Arabian beekeeping locations for infection by Paenibacillus larvae. Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and is the causative agent of American foulbrood disease. Larvae from samples suspected of infection were collected from different apiaries and homogenized in phosphate-buffered saline. Bacteria were isolated on MYPGP agar medium. Two bacterial isolates, ksuPL3 and ksuPL5 (16S rRNA GenBank accession numbers, KR780760 and KR780761, respectively), were subjected to molecular identification using P. larvae-specific primers. A BLAST sequence analysis revealed that the two isolates were P. larvae with more than 98% sequence identity. This detection of P. larvae in the indigenous honey bee is the first recorded incidence of this pathogen in Saudi Arabia. This study emphasizes the need for the relevant authorities to take immediate steps towards treating and limiting the spread of this disease throughout the country.