Paenibacillus larvae is a Gram-positive sporogenic bacterium that causes American Foulbrood (AFB). Five ERIC genotypes are described, which also differ phenotypically. In 2017–2019, P. larvae isolates were obtained during AFB outbreaks in Slovenia. Together with the isolates, we also obtained the corresponding epidemiological data. For the determination of P. larvae by bacteriological examination, we validated the MALDI TOF mass spectrometry method, thus speeding up and reducing the cost of the investigation. Two morphotypes of P. larvae colonies were observed. We found that the ERIC I genotype phenotypically mostly shows one morphotype – gray rough colonies, and the ERIC II genotype another – gray smooth or pigmented colonies. For the first time, ERIC-PCR was used for P. larvae typing in Slovenia and the frequency of ERIC genotypes could be determined. ERIC II was the predominant genotype (70,2 %). Some ERIC I isolates exhibited a slightly altered ERIC I banding pattern, thus 59 isolates were selected for the whole-genome sequencing (WGS). Despite showing non-classical ERIC I pattern, WGS-typing using the in silico MLST revealed that all of the investigated isolates belonged to the sequence type ST2. When comparing the whole-genome loci using the wgMLST analysis of the investigated isolates supplemented with all the publicly available P. larvae genomes, all Slovenian isolates fell within the ERIC I clade and phylogenetically clustered with the remaining ERIC I-ST2 genomes. Using molecular and phenotypic methods, we confirmed the presence of different morphotypes and genotypes of P. larvae in the same AFB zone or even in the same apiary. Results of the questionnaire statistically confirmed the presence of clinical signs characteristic for genotypes ERIC I and II, despite many other random factors that may affect the expression of AFB clinical picture and were not monitored in the scope of our study. In the genomes of P. larvae isolates, we identified the presence of gene clusters for the synthesis of inhibitory substances. Using the diffusion method, the ability to inhibit the growth of some Gram-positive bacteria and the yeast Malassezia pachydermatis was identified for individual ERIC I strains. The implemented diagnostic methods and study results represent an important contribution to the understanding of P. larvae genetic characteristics and AFB transmission routes.