The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.