Pasture decline is considered to be a serious challenge to agricultural productivity of subterranean clover across southern Australia. Root disease is a significant contributing factor to pasture decline. However, root disease assessments are generally carried out in the early part of the growing season and in areas predominantly sown to permanent pastures. For this reason, in spring 2004, a survey was undertaken to determine the severity of root disease in mature subterranean clover plants in pastures located in the wheatbelt of Western Australia. DNA-based soil assays were used to estimate population density in the soil of a variety of soil-borne pathogens known to commonly occur in the Mediterranean-type environments of southern Australia. The relationships between severity of disease on tap and lateral roots and root diameter, root length, nodulation, and total rainfall were determined. The survey showed, for the first time, that severe root disease is widespread in spring across the wheatbelt of Western Australia. There was a positive correlation between rainfall and tap root disease, and between tap root disease and average root diameter of the entire root system. Despite the high levels of root disease present across the sites, the DNA of most root disease pathogens assayed was detected in trace concentrations. Only Pythium Clade F showed high DNA concentrations in the soil. DNA concentrations in the soil, in particular for Phytophthora clandestina and Rhizoctonia solani AG 2.1 and AG 2.2, were higher in the smaller autumn sampling in 2006. This study suggests that the productivity of subterranean clover-based pastures is severely compromised by root rot diseases throughout the growing season in the wheatbelt of Western Australia.