American foul brood (AFB) disease is a deleterious bacterial disease worldwide, caused by spore forming bacterium Peanibacillus larvae that affects honeybee larvae and causes a significant decrease in the honeybee population. Following symptomatical and bacteriological approaches combined with 16S rDNA sequencing, an assessment has been made to evaluate the presence of AFB disease in North-West Pakistan as no record for bee-associated bacterial disease from Pakistan is available. A total of 1276 samples from 1520 bee colonies (15 apiaries) were collected, of which 476 samples (37.30%) were found with symptoms of AFB. Biochemical and 16S rDNA analysis indicated that all these farms have Peanibacillus larvae infection. It is concluded that the prevalence of AFB bacterial disease to such an extent in these regions of Pakistan will devastate the apicultural industries in a large scale across the country.