Accurate characterization of predator-prey linkages in agroecosystems is important prior to the implementation of conservation biological programs. The Russian wheat aphid, Diuraphis noxia (Hemiptera: Aphididae), is a significant pest of wheat and barley in the United States. This research utilized molecular gut-content analysis as a minimally disruptive technique to characterize the trophic connectivity between two spider species, Tetragnatha laboriosa Hentz 1850 and Pardosa sternalis (Thorell 1877), and D. noxia. We describe the development of species-specific primers that amplify a 227 bp fragment of D. noxia COI mtDNA to identify the frequency of predation under varying aphid densities and developmental stages of winter wheat. We tested the hypotheses that predation rates on D. noxia would be highest for both spider species at the greatest aphid infestation level in the aphid-resistant wheat cultivar plots and that densities of T. laboriosa would be highest at the highest aphid infestation level in the aphid-resistant cultivars. Despite short detection periods of prey DNA in the laboratory, 32% and 48% of field-collected T. laboriosa and P. sternalis spiders screened positive for D. noxia DNA, respectively. T. laboriosa densities were highest at the highest aphid infestation level. Aphid-resistant wheat cultivars did not impact predation rates or densities. Additionally, P. sternalis predation on D. noxia increased with increasing aphid infestation levels. Given the high predation rates on D. noxia and their association with increased aphid densities, both spider species represent important natural enemies within wheat agroecosystems, and further research is required to quantify their impact on aphid populations.