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Previously, we established that three skin-derived ketones including 6-methyl-5-hepten-2-one, acetophenone and geranyl acetone accounted for avoidance of zebra by tsetse flies. Here, we tested the hypothesis that these three ketones serve as biomarkers for tsetse flies to distinguish between non-preferred- and preferred-vertebrate hosts. We used coupled gas chromatography\/mass spectrometry to analyze and compare the skin volatile emissions of two non-preferred- (waterbuck and zebra) and four preferred- (buffalo, donkey, horse, warthog) vertebrate hosts in two wildlife parks in Kenya. We detected a total of 96 volatile organic compounds (VOCs) in the skin emissions composed mainly of aldehydes, ketones, alcohols, phenols and alkanes, which varied with the vertebrate host. Using random forest analysis, we found a weak correlation between the three skin-odor repellent ketones and non-preferred and preferred vertebrate hosts. However, we found that the three repellent ketones plus skin background odors may be more sensitive chemical signals for tsetse flies to discriminate vertebrate hosts. 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