Virus diseases can cause significant economic losses in agriculture and may pose biosecurity risks for crop production and trade. In 2010 a devastating disease of lucerne, named alfalfa dwarf disease (ADD) with an incidence greater than 70%, occurred in several commercial fields in Argentina and caused up to 30% annual yield and seed weight reduction. Next generation sequencing and field surveys of ADD-infected lucerne plants in Argentina revealed the presence of a new cytorhabdovirus, alfalfa dwarf virus (ADV), the (+) RNA viruses alfalfa mosaic virus (AMV), bean leafroll virus (BLRV) and alfalfa enamovirus 1 (AEV-1) and the DNA virus alfalfa leaf curl virus (ALCV). All these viruses appear to be transmissible by Aphis craccivora. Due to the economic impact of ADD in South America and its potential threat to the Australian lucerne industry, we developed diagnostic assays to determine the potential presence of ADD-associated viruses in Australia. In this study, lucerne leaves with virus-like symptoms were collected from seed crops in South Australia and fodder crops in Victoria and Queensland. A sensitive duplex RT-PCR assay was developed for simultaneous detection of ADV and AMV using a cloned non-infectious ADV RNA transcript as positive control. Similarly, the presence of BLRV and AEV-1 was determined by duplex RT-PCR and ALCV by PCR assay. None of the exotic viruses ADV, AEV-1 and ALCV were detected in any lucerne samples we tested in 2015/16. However, the endemic AMV and BLRV were detected in some samples, with a high incidence of AMV in symptomatic seed production paddocks. Symptoms in the collected samples varied widely and there was no obvious correlation between the symptoms observed in individual samples and presence of individual viruses. Future epidemiological studies in Argentina will assist in the development of a biosecurity plan to prevent ADD from occurring in Australia.