American foulbrood (AFB) is one of the most devastating infectious diseases of honey bees with a worldwide distribution. Based on clinical symptoms of AFB, the disease has been thought to have long existed in the Republic of Kosovo honey bee colonies. This passive surveillance study was intended to confirm the presence and determine the distribution of AFB in the Republic of Kosovo, based on the examination of clinical symptoms, rapid immunochromatographic tests, and microbiological and molecular identification of the etiological agent Paenibacillus larvae. From 2007 until 2019, official veterinary inspectors collected 2027 brood combs from a total of 373 apiaries clinically suspected as an AFB outbreak. Samples were collected in 26 out of 38 municipalities of the country predominantly during spring and autumn. P. larvae were isolated from 219 out of 373 apiaries, i.e., 58.71%. All samples with AFB clinical symptoms of the disease were positive in the immunochromatographic test. The presence of the AFB was demonstrated in 24 municipalities. Seventeen positive apiaries from different regions were randomly selected in the years 2016 to 2018 in order to characterize the isolates circulating most recently by ERIC genotyping. The presence of both ERIC I and II genotypes, as well as of a biotype with a profile slightly different from ERIC I and II, was observed. The results indicate a need for a change in approach in future control strategies and training programs for early diagnosis and improved management of apicultural practices.