The first cases of infection with the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae) in red foxes (Vulpes vulpes) from Portugal are described. Worms were collected from 1 fox (7 worms) in the north and from 2 foxes (10 worms) in the central region of the country. Partial molecular amplification of mitochondrial cythocrome c oxidase subunit 1 gene and sequencing revealed a 100% homology with T. callipaeda haplotype 1, which is the sequence type circulating in Europe. Data suggest that wildlife participate in maintaining this endemic infection in dog and cat populations from the studied areas. Furthermore, due to the zoonotic potential of T. callipaeda, there is a risk of infection in humans residing in areas where thelaziosis is present in wild and domestic animals.