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SUMMARY: Paecilomyces lilacinus is an agent for the potential biological control of soil nematodes. Arbitrarily primed PCR was used to fingerprint the genomes of 28 isolates of this fungus. Most (72%) of the isolates originated from soil of different regions of Brazil. Fourteen 10-mer oligonucleotide primers of arbitrary sequence revealed 293 scorable binary characters. Distinct genotypes were obtained for each isolate. Cluster analysis showed a high level of variability among these genotypes. The similarity among pairwise comparisons of the isolates varied from 84·3% to 7·6%, with a mean of 63·5%. No clearly defined phenetic groups were identified by cluster or multivariate analyses. No correlation with geographical origin or host was detected. In addition, PCR with four pairs of consensus tRNA gene primers was performed on a subsample of 12 P. lilacinus isolates, three P. farinosus isolates, two P. fumosoroseus isolates, and one isolate of P. amoenoroseus. An inferred phylogeny based on 112 binary characters obtained by tRNA-PCR showed a monophyletic group which contained most of the P. lilacinus isolates. In contrast, three isolates of P. farinosus were not in a monophyletic group under the inferred phylogeny. These results suggest that tRNA fingerprinting could provide a valuable tool which could be used to develop the molecular taxonomy of Paecilomyces, as morphological characteristics of asexual structures cannot entirely resolve species.