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This study was conducted to clarify the host specificity and the geographical distribution of Gasterophilus species (Diptera, Oestridae) in the Serengeti ecosystem. A total of 317 larvae were recovered from two common zebras (Equus quagga, formerly Equus burchellii) in Maswa Game Reserve, and 58 larvae were recovered from an African lion (Panthera leo) in the Serengeti National Park. The study emphasizes the rare occurrence of Gasterophilus sp. in lions, shedding light on the broader life cycle and physiological implications for hosts. Genetic analysis of cox2 genes from Gasterophilus species, sourced from a single geographic location, reveals significant genetic distinctions and host specificity. This study reports the first case of G. intestinalis infestation in an African lion in the Serengeti ecosystem, extending its known range from zebras and other equids, and highlighting ecological and veterinary implications. This unusual prey-predator transmission highlights the value of molecular taxonomic tools in clarifying host-parasite dynamics and guiding targeted conservation strategies.