© 2017 Blackwell Verlag GmbH Screening of field isolates of Pestalotiopsis sp. from strawberry leaf blight in Thailand identified 56 carbendazim-resistant isolates. Of 56 isolates, 39 highly resistant (HR) phenotypes grew well on PDA amended with carbendazim even at ≥500 mg/L. Isolates with carbendazim-resistant phenotype had a conspicuous mutation at particular sites in the beta-tubulin (β-tubulin) gene sequence. A β-tubulin encoding gene from this pathogen was cloned and sequenced. Analysis of the β-tubulin gene in highly resistant (HR) isolates showed a substitution at codon 79 and 102 from serine (AGT) to lysine (AAA) and valine (GTA) to alanine (GCA), respectively. The detection of such point mutations in the β-tubulin gene allows the rapid screening to detect carbendazim-resistant isolates in the field.