A new species of the spider genus Loxosceles, L. mrazig sp. n., found in Tunisia is described and illustrated. The male bulb shows a high degree of morphological similarity to that of L. gaucho from Brazil, but the proportions of the palpal segments and the general colouration of the body reveal significant differences between the two species. A distance analysis of the sequences of the mitochondrial gene cox1 reveals that the specimen from Tunisia shows high genetic distance from L. gaucho (more than 20%). The American species L. gaucho and L. laeta form a sister group to the Mediterranean representatives (L. rufescens and the Tunisian specimen).