Restriction fragment length polymorphism analyses of polymerase chain reaction (PCR) amplified DNA were used to distinguish Diaporthe phaseolorum and Phomopsis longicolla isolates from other soybean fungal pathogens. Primers made to the conserved sequences of nuclear ribosomal DNA amplified the internal transcribed spacer (ITS) regions of D. phaseolorum var. meridionalis and P. longicolla. The PCR products were cloned and then sequenced. Specific-primers, Phom.I and Phom.II, were designed from the polymorphic regions of D. phaseolorum and P. longicolla isolates from soybean to distinguish them from other soybean fungal pathogens. These ITS-derived primers amplified a 337-bp-specific DNA fragment from P. longicolla, D. phaseolorum var. meridionalis, D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and Phomopsis spp. from 20 different hosts. No amplified product was observed using DNA of seven other soybean fungal pathogens or soybean DNA. The detection limit of PCR using primers Phom.I and Phom.II was 2.5 × 10-7 dilution of fungal DNA extracted from samples of 10 pooled seeds and as low as a 1:15 (Phomopsis:soybean) ratio when using 10 ng of DNA per μl from each P. longicolla and soybean. PCR did not produce products using primers Phom.I and Phom.II with DNA extracted from noninfected seeds, but specific bands were observed from samples of 10 pooled seeds and from individually infected seeds. A specific band was observed as well from DNA extracts of tissue samples from symptomless plants inoculated with P. longicolla and D. phaseolorum var. sojae.