A couple of different members of the coleopteran genus Otiorhynchus (Coleoptera: Curculionidae) are becoming increasingly important as pests of nursery and ornamental plants in global horticulture. Although adult weevils are morphologically distinguishable by skilled personnel, high potential for misidentification is given for cryptic larval stages. For developing and applying efficient pest management strategies the determination of the respective species is however a prerequisite, because each species may have a different phenology or a varying susceptibility to pesticides. Here, we report on the development of a diagnostic polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for differentiation among 16 Otiorhynchus and seven other weevil species independent of their developmental stage. An approximately 780-bp fragment of the mitochondrial cytochrome oxidase subunit II was amplified and subsequently digested with at most four restriction enzymes generating species-specific fragment patterns. The assay was validated on a total of 127 individuals and the obtained fragment patterns correctly identified 23 different weevil species. The PCR-RFLP method reported here is cost-effective, robust, and fast and could be used in the future by plant protection services for diagnostic purposes.