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In clinical practice, mixed-species malaria infections are often not detected by light microscopy (LM) or rapid diagnostic test, as a low number of parasites of one species may occur. Here, we report the case of an 8-year-old girl migrating with her family from Afghanistan with a two-species mixed infection with Plasmodium vivax and Plasmodium ovale. This case demonstrates the significance of molecular testing in the detection of mixed-species malaria infections and highlights the importance of a detailed data analysis during the medical validation procedure to prevent underestimation of mixed-species infections. To our knowledge, this is the first case report of a two-species mixed infection comprising both P. vivax and P. ovale confirmed by LM and different real-time PCR approaches.