A DNA fragment apparently unique to Verticillium dahliae was found by comparing RAPD profiles of V. dahliae to those of other closely related fungi. RAPD analyses were performed on eight V. dahliae, six V. albo-atrum and three V. tricorpus isolates to identify DNA sequences specific to V. dahliae. RAPD primer E20 (AACGGTGACC) yielded a 567 bp band shared only by V. dahliae isolates. This band from isolate V14 was cloned and sequenced. No significant sequence similarity was found between this amplicon and any other nucleic acid sequence in the databases. Based on the sequence information, a pair of PCR primers, VDS1 (5′-CACATTCAGTTCAGGAGACGGA-3′) and VDS2 (5′-CCTTCTACTGGAGTATTTCGG-3′) was designed. PCR tests showed that VDS1 and VDS2 amplified the expected fragment of DNA from 62 V. dahliae isolates from diverse hosts and geographical origins, but not from any other sources of DNA tested, including DNA from the most closely related species, V. albo-atrum. Southern blot analysis showed that the PCR product specifically hybridized to V. dahliae genomic DNA. An internal control was constructed for competitive PCR and used to develop a detection and quantification assay for V. dahliae, for which the detection limit was determined.