The species of the genus Orius Wolff are well known as generalist predators able to control pest outbreaks in several agroecosystems. Correct species identification can be problematic given their similarities, particularly in the immature stage. A pair of primers previously designed from the internal transcribed spacer-1 (ITS-1) region was able to discriminate between seven Orius species commonly present in Mediterranean vegetable crops (Orius majusculus, Orius laevigatus, Orius minutus, Orius laticollis, Orius horvathi, Orius albidipennis and Orius niger), as well as to correctly identify O. majusculus and O. laevigatus from commercial colonies. This molecular tool was used for the discrimination of Orius spp. present in two lettuce crops, as well as in a Lobularia maritima flower margin in northeast Spain in 2009 and 2010. Molecular analyses revealed that O. laevigatus, O. majusculus and O. niger were the most common species present in both lettuce plots and the L. maritima border, although there was some variation depending on the plant and year. This molecular tool permits unambiguous identification of these species and allows proper implementation of biological control programs based on conservation. (C) 2013 Elsevier Inc. All rights reserved.