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It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles.