Bacteriophage endolysins present enormous biotechnological potentials and have been successfully used to control and detect bacterial pathogens. Endolysins targeting Gram-positive bacteria are modular, displaying a cell binding (CBD) and an enzymatically active domain. The CBD of phage endolysins are recognized by their high specificity and host affinity, characteristics that make them promising diagnostic tools. No CBD able to bind Paenibacillus larvae has been identified so far. P. larvae is a Gram-positive spore forming bacteria that causes the American Foulbrood. This highly contagious infection leads to honeybee larvae sepsis and death, resulting in an adverse impact on pollination and on the beekeeping industry. In this work, the first CBD targeting P. larvae was identified and its core binding sequence was investigated. Moreover, it was shown that the domain is highly specific, targeting exclusively P. larvae cells from all ERIC genotypes. The identification of such a domain represents a step forward for the development of effective methods to detect and control this pathogen.