Aerial insectivores are highly mobile predators that feed on diverse prey items that have highly variable distributions. As such, investigating the diet, prey selection and prey availability of aerial insectivores can be challenging. In this study, we used an integrated DNA barcoding method to investigate the diet and food supply of Barn Swallows, an aerial insectivore whose North American population has declined over the past 40 yr. We tested the hypotheses that Barn Swallows are generalist insectivores when provisioning their young and select prey based on size. We predicted that the diets of nestlings would contain a range of insect taxa but would be biased towards large prey items and that the diet of nestlings would change as prey availability changed. We collected insects using Malaise traps at 10 breeding sites and identified specimens using standard DNA barcoding. The sequences from these insect specimens were used to create a custom reference database of prey species and their relative sizes for our study area. We identified insect prey items from nestling fecal samples by using high-throughput DNA sequencing and comparing the sequences to our custom reference database. Barn Swallows fed nestlings prey items from 130 families representing 13 orders but showed selection for larger prey items that were predominantly from 7 dipteran families. Nestling diet varied both within and between breeding seasons as well as between breeding sites. This dietary flexibility suggests that Barn Swallows are able to adjust their provisioning to changing prey availability on the breeding grounds when feeding their nestlings. Our study demonstrates the utility of custom reference databases for linking the abundance and size of insect prey in the habitat with prey consumed when employing molecular methods for dietary analysis.