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Resistance to pyraclostrobin due to a single nucleotide polymorphism at 143rd amino acid position on the cytochrome b gene has been a major source of concern in red pepper field infected by anthracnose in Korea. Therefore, this study investigated the response of 24 isolates of <i>C. acutatum</i> and <i>C. gloeosporioides</i> isolated from anthracnose infected red pepper fruits using agar dilution method and other molecular techniques such as cytochrome b gene sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific polymerase chain reaction (PCR). The result showed that four isolates were resistant to pyraclostrobin on agar dilution method and possessed GCT (alanine) codon at 143rd amino acid position, whereas the sensitive isolates possessed GGT (glycine). Furthermore, this study illustrated the difference in the cytochrome b gene structure of <i>C. acutatum</i> and <i>C. gloeosporioides</i>. The use of cDNA in this study suggested that the primer Cacytb-P2 can amplify the cytochrome b gene of both <i>C. acutatum</i> and <i>C. gloeosporioides</i> despite the presence of various introns in the cytochrome b gene structure of <i>C. gloeosporioides</i>. The use of allele-specific PCR and PCR-RFLP provided clear difference between the resistant and sensitive isolates. The application of molecular technique in the evaluation of the resistance status of anthracnose pathogen in red pepper provided rapid, reliable, and accurate results that can be helpful in the early adoption of fungicide-resistant management strategies for the strobilurins in the field.

<i>Colletotrichum</i> species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of <i>Colletotrichum acutatum s. lat.</i> and <i>Colletotrichum gloeosporioides s. lat.</i> The result of the ADM showed that <i>C. gloeosporioides s. lat.</i> was classified into sensitive and resistant isolates to benomyl while <i>C. acutatum s. lat.</i> was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of <i>C. gloeosporioides s. lat.</i> Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

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