Simple SummaryGastrointestinal nematode (GIN) infections constitute a serious threat to the cattle industry, and anthelmintics have been widely used to control them. As a result, resistance to broad-spectrum drugs is now a worldwide issue. In the present study, we evaluated the resistance status of GINs to fenbendazole (FBZ) in cattle of an Ecuadorian farm with a known history of broad-spectrum anthelmintic usage. We found that the GIN population was susceptible to FBZ andCooperiaspp. was the most prevalent genus before and after treatment. Although the reduction in egg count inCooperiaspp. was satisfactory (97.96%), we detected a phenylalanine-to-tyrosine substitution at codon 200 (F200Y) in the β-tubulin 1 gen in 43% of the pooled larva coproculture after treatment. The presence of F200Y suggests the existence of resistance in the early stages. Our findings highlight the need to implement alternative control strategies for parasitic infections besides the usage of broad-spectrum anthelmintics.AbstractAnthelmintic resistance among GINs in cattle is a worldwide issue. Identifying the early signs of anthelmintic resistance (AR) is necessary to sustainably manage bovine parasitic infections. This study aimed to evaluate the resistance status of bovine parasitic nematodes against FBZ on a farm with a known history of broad-spectrum anthelmintic usage in Ecuador. FBZ efficacy was analyzed using a fecal egg count reduction test (FECR test) and β-tubulin 1 mutation identification inCooperiaspp., the dominant nematode parasite identified before and after treatment. According to the FECR test, the nematode population was susceptible to FBZ. After amplifying and cloning the β-tubulin 1 ofCooperiaspp., an F200Y mutation was found in 43% of the pooled larva coproculture after treatment. This study reports, for the first time, the presence of F200Y resistance-conferring mutation inCooperiaspp. in Ecuador. Although the nematode population was phenotypically susceptible to FBZ, the presence of F200Y suggests the existence of resistance in the early stages. Our findings highlight the need to implement alternative control strategies for parasitic infections besides broad-spectrum anthelmintics.