70 strains of Aspergillus ochraceus mainly isolated from Brazilian coffee related sources were investigated for genetic relatedness using automated laser fluorescence analysis of AFLP fragments. Cluster analysis of fingerprints revealed a very close relationship among most of the strains. Based on these results, a sub-set of characteristic A. ochraceus strains was chosen for the detection of marker sequences. These sequences were obtained from silver stained AFLPs separated on polyacrylamide gels. A number of bands characteristic for A. ochraceus were detected and cut out from the gels. DNA was reamplified, cloned and fragments were sequenced. Based on these sequences a set of SCAR PCR-primers was constructed. PCRs were optimised for specificity and subsequently tested against a panel of Aspergillus species. Using this approach a PCR specific for Aspergillus ochraceus was developed.