BACKGROUND Rodenticide resistance to anticoagulants in Rattus norvegicus will lead to increased difficulties in combating these pest animals. Here, the authors present the results of a survey in the Netherlands where tissue samples and droppings were tested using a newly developed TaqMan PCR test for genotypic variation at codon 139 in the Vkorc1 gene associated with anticoagulant rodenticide resistance. Test results are linked to results of a questionnaire that was conducted among pest controllers. RESULTS Genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus can be encountered in many parts of the Netherlands. In 34/61 rat tails, a genotype was found that is linked to anticoagulant rodenticide resistance (56%). In droppings, 42/169 samples (25%) showed a resistance-mediating genotype. In addition, indications of a clear genetic substructure in the Netherlands were found. In some regions, only resistance-mediating genotypes were found, corroborating results from the questionnaire in which pest controllers indicated they suspected resistance to anticoagulant rodenticides. CONCLUSIONS This is the first study to demonstrate the presence of multiple genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus in the Netherlands. As rodenticides should keep their efficacy because they are a last resort in rodent management, more studies are urgently needed that link specific genetic mutations to the efficacy of active substances.