The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.