The fungal species Fusarium oxysporum, F. graminearum, F. acuminatum, F. verticilliodes, F. equiseti, F. proliferatum, F. solani, F. sporotrichioides and Rhizoctonia solani are soil-borne pathogens responsible for yield loss of multiple crops and are known causal agents of important diseases in soybean, including root rots and seedling diseases. The early and accurate identification of these pathogens represents a decisive step in managing these diseases. Therefore, we have designed highly species-specific primer-probe assays for the identification of these soil-borne pathogens. The primers and probes were designed based on the intergenic spacer ribosomal RNA (IGS) and translation elongation factor alpha gene (EF1?). The specificity of the designed panel was tested against 21 non-target pathogenic fungal species. All the designed sets showed early amplification using genomic DNA of the target species and were specific enough to not amplify all non-target species. The designed primer-probe sets were also successfully used in identifying and quantifying target pathogens in roots and soil samples collected from in soybean fields across different states in the US. We have developed a precise set of primer and probes to detect and quantify major soil-borne pathogens in fields, which could guide decision-making in disease management by providing early detection of these pathogens in the soil.