Simple SummaryHaemonchus contortusis well known as the most harmful gastrointestinal nematode in ruminants, causing serious economic losses worldwide. The overuse of chemical insecticides (e.g., ivermectin) has caused gastrointestinal nematode resistance to develop and spread, resulting in failure in controlling gastrointestinal nematode spread. At the start of our study, four varieties ofH. contortuswere obtained from diverse regions of China. This study investigated the drug resistance ofH. contortusisolates to ivermectin. Additionally, the changes in theP-gpgene transcription levels of third-stage larvae under ivermectin stress at different time points were measured. The results indicated that the YCHc-022 strain was sensitive to ivermectin, but the WSHc-001, WSHc-003, and CYHHc-136 strains displayed different degrees of resistance to ivermectin, in comparison with the ivermectin-sensitiveH. contortusfound in Australia. Results from the third-stage larvae ofH. contortusin response to ivermectin stress revealed a relation between theP-gpgenes and the drug resistance ofH. contortusto ivermectin; however, it was not suitable for use as an international molecular marker. Our findings are beneficial for screening molecular markers ofH. contortusresistance to ivermectin and offer a novel perspective for detecting the drug resistance of gastrointestinal nematodes.AbstractHaemonchus contortusposes a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment ofH. contortusparasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains ofH. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes inP-gpexpression levels can be used to analyze the resistance ofH. contortusto ivermectin. This study aimed to analyze the effects of ivermectin onP-gpexpression inH. contortusL3 larvae isolated from China and to evaluate whether changes inP-gpexpression levels can be used to analyze resistantH. contortusstrains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression ofP-gp11 (p< 0.01) compared with sensitive strains from elsewhere, whereas the expressions ofP-gp2 andP-gp9.1 were downregulated (p< 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-inducedP-gpexpression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that usingP-gpto determine ivermectin resistance inH. contortusstrains from different geographic environments can yield different results.