American Foulbrood Disease (AFB) is considered one of the most virulent bacterial diseases of honeybee (Apis mellifera); it has a vital negative impact on the beekeeping industry worldwide. This work includes the subsequent diagnosis including; chemical, microbiological procedure for detection of Paenibacillus larvae. During the spring and the summer of 2009, fifty-six honey and larval samples were collected from 56 inspected honeybees colonies located in 53 apiaries representing beekeeping all over Jordan. The samples were examined to assess the presence of Paenibacillus larvae in honey samples by using both chemical and microbiological methods. Honey bees field diagnosis procedures were considered to be the first diagnosis step for AFB, which can be done by the beekeepers themselves. The total of 57 (honey brood and brood nest honey) from different regions of Jordan was inspected to carry P. larvae spores with 35%. The percent referred to the ability of Jordanian beekeepers to ascertained AFB symptoms. American foulbrood exists in Jordan with different distribution depending on the environmental condition. This pathology can be best detected by isolation of Paenibacillus larvae from Brood-nest honey samples.