The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the beta-tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the beta-tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding beta-tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2–99.6% at the nucleotide and 98.7–100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from individuals of Cc. nassatus, Cy. coronatum, Cy. pateratum, and Cy. catinatum of a BZ-susceptible one showed differing amino acids in 11 positions. The commonness of a phenylalanine to tyrosine mutation at position 167 in all the 6 cyathostomin species isolated from a BZ-resistant population suggests its involvement in the molecular mechanism in BZ resistance.