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Simple Summary: Detection dogs and their handlers must be a bonded team. Changing a dog's handler, which occurs in certain organisations or through change of ownership, may generate team conflict and reduce detection performance. Through testing dogs at detection tasks with a familiar and unfamiliar handler, we found that dogs scored higher for detection accuracy with their familiar handler. The dogs were also less distracted with their familiar handler. These results suggest that changing a dog's handler influences both their detection accuracy and behaviours. This may impact how working dogs are managed and their welfare. Dog-handler relationships can directly impact team success. Changing a dog's handler may therefore compromise detection performance. However, there are currently few studies which support this. This research explored the performance and behavioural impact of changing a dog's handler. Nine dogs trained at scent detection were accuracy tested with a familiar and unfamiliar handler. Both handlers were female with similar dog handling experience. The dogs were tested along brick lines containing target, non-target, and control samples. Testing was separated into four sessions, with each session having 36 samples. The dogs' accuracy scores were then calculated and testing footage behaviour coded. The dogs had significantly higher sensitivity (p = 0.045) and negative predictive value (NPV) (p = 0.041) scores when handled by the familiar handler. With the unfamiliar handler the dogs performed more stress-related behaviours, and were distracted for a higher proportion of time (p = 0.012). Time spent distracted was negatively correlated to detection performance (correlation = −0.923, p < 0.001). With the unfamiliar handler the dogs' performance did not improve throughout testing (p = 0.553). This research demonstrates how these dogs' detection performances were impacted by changing handlers. Future research is required to determine if professional dog-handler teams are impacted similarly. [ABSTRACT FROM AUTHOR]