Definition of the genus Calliptamus (Orthoptera: Acrididae) has generated many taxonomic debates. Even now, the existence of different geographical morphs hinders species determination, particularly as concerns females and larvae. Some of these species are observed in southern France and are recognized as potential pests. To circumvent problems of species identification in ecological surveys, we developed a single multiplex PCR method based on mitochondrial Cytochrome Oxydase I diagnostic polymorphisms to differentiate between the four species, Calliptamus italicus, C. wattenwylianus, C. siciliae and C. barbarus, in southern regions of France.