Abstract: Orius majusculus (Hemiptera: Anthocoridae) is an important generalist predatorknown to feed on two of the main pests in lettuce crops, Frankliniella occidentalis(Thysanoptera: Thripidae) and Nasonovia ribisnigri (Hemiptera: Aphididae). Diagnosticmolecular gut-content analysis has been conducted to study predation of O. majusculus on thesepests, as well as on the springtails of the genus Entomobrya sp. (Collembola), the most abundantnon-pest prey in lettuce crops of the studied area. Two pairs of specific primers were designed forN. ribisnigri and F. occidentalis, amplifying 331 bp and 277 bp fragments of the mitochondrialCytochrome c Oxidase I (COI) region, respectively. Collembola detection, was conducted with apair of a group-specific primers previously designed that amplify an amplicon of 177 bp of the18S rDNA region. Prey was detected in 100% of predators immediately after prey consumption(t = 0h). PCR analysis of O. majusculus collected in the field revealed a strong relationshipbetween prey density in the crop and consumption. Sixty per cent of these field-collectedO. majusculus were positive for N. ribisnigri in spring (when this pest species was present) andup to 40% were positive for F. occidentalis in summer (when the density of this pest was thehighest). Collembolan detection ranged from 9.4% to 28.2% depending on the season andpredator stage. A higher proportion of immature O. majusculus ingested more than one preyspecies simultaneously compared to adults of the same predator species. These findings suggestthat O. majusculus is an important predator for the control of both lettuce pests, being springtailsan important alternative prey, which could maintain O. majusculus populations when both pestspecies are not present in the crop or are not abundant enough.