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Trichostrongyles are gastrointestinal parasites that occur globally and can cause subclinical to severe, sometimes life-threatening, infections in ruminants, particularly young animals. Benzimidazoles (BZ) are commonly used for the treatment of gastrointestinal parasites in ruminants. Increasing spread of worm populations with anthelmintics resistance has been reported and is considered a consequence of highly frequent and longstanding use of anthelmintics. To obtain initial information regarding the occurrence of putatively BZ-resistant Nigerian Haemonchus populations, screening based on the molecular analysis of BZ-resistance-associated β-tubulin isotype 1 gene sequence polymorphisms was undertaken. Genomic DNA was isolated from pooled adult Haemonchus sp. from 35 animals from each of the six states of southwestern Nigeria. Sequencing of internal transcribed spacer 2 (ITS-2) and external transcribed spacer (ETS) regions was used to determine the Haemonchus species. Pyrosequencing assays were used for detection of single-nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 genes of the worms at codons 200 and 167 (TTC/TAC) or 198 (GAA/GCA). Exclusively, Haemonchus placei was detected and allele frequencies obtained at all three positions showed no evidence for the presence of resistance-related alleles. For Lagos State, pools of 10 worms from 30 different animals were analyzed separately for the codon 200 SNP, successfully excluding the presence of resistance-associated SNPs in very low frequencies. These positive findings, showing absence of elevated frequencies of BZ-resistance-associated β-tubulin alleles, have considerable significance since it suggests that farmers can still rely on the efficacy of this important drug class when used for controlling trichostrongyle infections in cattle in Nigeria.