A single-step multiple-target (multiplex) reverse transcription-PCR (RT-PCR) was developed for the simultaneous detection and differentiation of three economically important viruses of the honeybee Apis mellifera L.: Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV) and Sacbrood virus (SBV). Three compatible sets of primers, specific for each virus, were designed in conserved regions of the viral genomes for use in a one-step (single tube) RT-PCR assay. The individual RT-PCR assays and the combined multiplex assay were optimized for highest sensitivity and specificity. The multiplex RT-PCR assay was tested on field samples collected from Austrian honeybee colonies. All three viruses were detected, and their identity was confirmed by sequencing of the PCR products. The described multiplex RT-PCR proved to be an accurate tool for rapid simultaneous detection of ABPV, BQCV and SBV directly in honeybee specimens.