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Anthelmintic resistance has emerged as an important problem in animal industries. Understanding resistance mechanisms, especially against macrocyclic lactones (MLs), is the first step in developing better diagnostic tools. Effects of several MLs including ivermectins and milbemycins were tested using two well established in vitro assays: the larval development assay (LDA) and the larval migration inhibition assay (LMIA). These were performed on free-living stages of susceptible and ML-resistant isolates of three trichostrongyloid nematode species of sheep. In general, dose response curves shifted to the right in the resistant isolates. Data showed that resistance was present to ivermectin and its two components suggesting that both components contribute to action and resistance. There were no consistent patterns of potency and resistance of the tested substances for the different isolates in the LDA except that moxidectin (MOX) tended to have lower resistance ratios than ivermectin (IVM). MOX was the most potent inhibitor in the LMIA in susceptible Haemonchus contortus while being less potent in Trichostrongylus colubriformis and particularly in Ostertagia circumcincta. MOX showed high resistance ratios in the LMIA in all three species. Based on these results, resistance to MOX has unique characteristics and the LMIA may perform better in detecting resistance to MOX in these parasite species.