Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.