Molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, random amplification of polymorphic DNA (RAPD) fingerprinting, and DNA sequencing and database comparison, were employed to identify fungi isolated from field-grown cotton plants (Gossypium hirsutum L.). DNA fragments of between 510 and 590 bp, representing the two rDNA (rDNA) internal transcribed spacers (ITS1 and ITS2) and the intervening 5.8S rRNA gene, were amplified from the fungi with eukaryotic consensus primers. Subsequent digestion with the restriction endonucleases AluI, CfoI, HaeIII, HinfI and HpaII enabled the allotment of all 57 isolates to 13 different groups. Restriction analysis was supported by RAPD–PCR analysis of multiple isolates and rDNA sequencing of representative fungi from each group. Sequence alignment and comparison with rDNA sequences of other fungi available in GenBank allowed for putative identification of three different taxa of Fusarium, two taxa each of Cladosporium, Diaporthe and Nectria, and one taxon each of Alternaria, Ampelomyces, Bartalinia, Phaeosphaeria and Rhizoctonia. Many of the stem-colonising fungi identified in this study are either pathogenic on cotton or have elsewhere been found to act as biocontrol agents.