Field reports of anthelmintic resistance against the widely-used macrocyclic lactones (ML) in gastrointestinal nematodes (GIN) of cattle have appeared in NW-Europe in recent years. Objective: The aim of this study was to assess the efficacy of ivermectin (IVM) against field infections with GIN in Danish cattle. In addition, we evaluated a novel quantitative (q) real-time PCR assay for accurate identification of surviving nematode species after treatment. Methods: Six farms were selected based on mean faecal egg counts (FEC) of ≥ 100 nematode eggs per gram in first season grazing heifers. All selected farms had a history of use of ML. In each farm, 20 heifers were selected for faecal egg count reduction test (FECRT) and individual weights were measured using a girth-tape. Ten animals were treated (s.c.) with 0.2 mg IVM/kg body weight and ten animals were left untreated as controls. FEC were investigated in all animals at the day of treatment (0) and at days 14 and 21 post-treatment (p.t). In parallel, L3 were cultured from pooled faeces from all animals in each group. DNA isolated from pooled L3 was analysed by a qPCR quantifying copies of the second internal transcribed spacer (ITS2) specific for Ostertagia ostertagi and Cooperia oncophora. FEC reduction percentages (FECR%) in IVM-treated groups were calculated following WAAVP guidelines including FECs of the control group. Reduced efficacy was defined when FECR% ≤ 95% and lower confidence interval [CI] ≤ 90%. Results: At day 14 p.t., FECR% of IVM treatments in all six farms varied from 75% to 96% (lower CI ranging from 39% to 91%). At day 21 p.t., IVM treatments showed a reduced efficacy in all farms, with FECR% varying from 45% to 91% (lower CI 95% ranging from -34% to 65%). At day 0, ITS2 copies from O. ostertagi and C. oncophora were indentified in larval cultures from treated groups in all six farms. At day 14 p.t. in treated animals, O. ostertagi L3 were found in 1 farm and C. oncophora L3 in 3 farms. On day 21 p.t., O. ostertagi L3 and C. oncophora L3 were detected in treated animals of 2 and 4 farms, respectively. Discussion: Reduced field efficacy of IVM was confirmed by FECRT in 5 out of 6 farms at day 14 p.t. and in all six farms at day 21 p.t. The qPCR was able to identify O. ostertagi and C. oncophora populations surviving IVM treatment. This highlights the need of monitoring the efficacy of anthelmintic treatments in cattle using sensitive methods.