Background: Reliable information on host use by arthropod vectors is required to study pathogen transmission ecology and to predict disease risk. Direct observation of host use is often difficult or impossible and indirect methods are therefore necessary. However, the reliability of currently available methods to identify the last host of blood-feeding arthropods has not been evaluated, and may be particularly problematic for ticks because host blood has been digested at capture. Biases in host detection may lead to erroneous conclusions on both vector ecology and pathogen circulation.Methods: Here, we experimentally tested for biases in host detection using the generalist three-host tick Ixodes ricinus as a model system. We fed ticks using an artificial feeding system and amplified blood meal traces post-moult (i.e., in the succeeding unfed life stage) via both a quantitative real-time polymerase chain reaction assay and a reverse line blotting method. We then experimentally tested for three types of biases in host detection: 1) time post-moult, 2) tick life stage and 3) host type (non-nucleated mammal blood versus nucleated avian blood), and compared these biases between the two molecular methods.Results: Our results show that all three factors can influence host detection in ticks but not necessarily in the expected way. Although host detection rates decreased with time post-moult, mammal blood tended to be more readily detected than bird blood. Tick life stage was also an important factor; detection was higher in nymphs than in adults and, in some cases, remnants from both larval and nymphal blood meals could be detected in the adult stage. These biases were similar for the two detection techniques.Conclusions: We show that different factors associated with questing ticks may influence our ability to correctly infer previous host use and that these factors may bias inferences from field-based studies. As these biases may be common to other vector-borne disease systems, their implications for our understanding of vector ecology and disease transmission require more explicit consideration.