Background: Benzimidazole (BZ) resistance in the gastrointestinal nematode Haemonchus contortus is a major constraint to sheep production worldwide. However, data on the prevalence and molecular mechanisms of resistance in Yili Prefecture, Xinjiang—a key livestock region in Northwestern China—remain limited. This study aimed to determine the frequency of BZ resistance-associated single nucleotide polymorphisms (SNPs) in H. contortus populations from Zhaosu and Tekesi counties. Methods: Adult male worms (n = 150) were collected from naturally infected sheep at local abattoirs. Species identity was confirmed morphologically by sequencing the internal transcribed spacer 2 (ITS-2) region. A 385 bp fragment of the isotype-1 β-tubulin gene was amplified and sequenced to detect SNPs at codons 167 (F167Y), 198 (E198A), and 200 (F200Y). Results: The F167Y mutation was absent in all individuals. In contrast, the E198A mutation occurred at exceptionally high frequencies, with resistant allele frequencies (RAF) of 64.7% in Zhaosu and 52.7% in Tekesi. The F200Y mutation showed clear geographical variation: it remained low in Zhaosu (RAF = 9.3%) but was substantially higher in Tekesi (RAF = 33.3%). Haplotype analysis revealed that resistance in Zhaosu was driven primarily by the E198A mutation, whereas the Tekesi population exhibited complex patterns of co-selection of both E198A and F200Y, with a high proportion of double-heterozygous individuals (29.3%). Conclusions: This study provides comprehensive molecular evidence of severe BZ resistance in H. contortus populations from Zhaosu and Tekesi counties, Yili Prefecture. The marked predominance of the E198A mutation, together with the emergence of multi-locus resistance in Tekesi, indicates a rapid escalation of resistance beyond historical levels. These findings suggest that benzimidazoles are likely ineffective in this region and highlight the urgent need to revise local parasite control strategies.

Benzimidazole (BZ) resistance in Haemonchus contortus constrains sheep production worldwide. Northwestern Xinjiang is an important livestock region, yet a parallel comparison of isotype-1 β-tubulin genotypes at the three canonical BZ-resistance loci has not been reported for Yining and Tacheng. Between July and August 2025, 148 adult male worms (77 from Yining and 71 from Tacheng) were collected from sheep processed at local abattoirs. Adult males were identified morphologically by the asymmetrical dorsal ray and the organisation of the bursal rays; females showed a barber-pole appearance. No other abomasal nematode species were observed in the examined material. Species identity of the 148 genotyped males was confirmed by ITS-2 PCR, with Sanger sequencing of 22 amplicons. Remaining males were identified by morphology only and were not subjected to molecular identification. A fragment of the isotype-1 β-tubulin gene spanning codons 167, 198 and 200 was amplified and Sanger-sequenced. Genotypes were coded SS, rS and rr. F167Y was absent from all worms. The two samples differed at E198A and F200Y. The Tacheng sample contained only heterozygous (rS) genotypes at these codons, with substitution-allele frequencies of 11.3% (E198A) and 13.4% (F200Y); SS genotypes predominated. The Yining sample contained rr genotypes at both codons, with higher substitution-allele frequencies (44.8% for E198A and 20.1% for F200Y) and more frequent co-occurrence of non-SS genotypes at the two codons. These findings provide a side-by-side molecular comparison of the two areas. Because sampling was abattoir-based, phenotypic assays were not performed, and individual treatment histories were unavailable, the results should not be read as confirmed clinical resistance or as a basis for region-specific drug protocols. They provide a baseline for further surveillance and for field efficacy testing of benzimidazoles in northern Xinjiang.