Given the considerable economic loss to beekeepers worldwide and the possible public health implications related to the presence of antibiotics in honey, an American Foulbrood (AFB) monitoring/prevention program for Paenibacillus larvae is regarded as essential. This study investigates the occurrence and distribution of P. larvae genotypes in honey and brood combs from Apulia (Italy). Genotyping of P. larvae isolates using ERIC-PCR generated a total of four different ERIC banding patterns (ERIC-A, ERIC-B, ERIC-C, ERIC-D), including fragments ranging from 200 to 3000 bp. Considering that the genotype has an influence on P. larvae infections and multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing the type and speed of the development of clinical symptoms, the findings of the present study could be helpful for training veterinarians, bee inspector's extension staff, and beekeepers, thus improving the detection of AFB infections in the field.