Development of specific DNA probes targeting rDNA internal transcribed spacer (ITS-1 or -2) sequences is described for the detection of strains representing uninucleate and binucleate Rhizoctonia spp. and Suillus bovinus. Discriminatory taxon/species-specific target sequences were identified following full length ITS sequence alignment of the test fungal sequences and those of other root associated pathogenic or mycorrhizal fungi. Both long (124–151 bp) and shorter (20–25 bp) probes were generated for assessment in Southern dot blot and liquid hybridization ITS capture-fragment length polymorphism assays. Fungal genomic DNA was presented as the target in dot blot protocols using the longer DIG (digoxigenin) labelled probes whilst the shorter 3′ biotin-labelled oligonucleotide probes were hybridized to PCR amplified full length ITS (ITS1-5.8S-ITS2) in both dot blot and liquid hybridization assays. The optimal hybridization temperatures for dot blot detection also produced maximal target specific signals in the liquid hybridization protocol. The latter protocol was found to be more discriminatory as target fungi were detected on the basis of combined probe hybridization-ITS capture and 5′ Cy-5 labelled ITS length polymorphism analysis (±5 bp) following denaturing sequencing gel electrophoresis in a ALFexpress DNA sequencer.