This dataset contains locations of worker bumblebees of five species (Bombus terrestris, B. lapidarius, B. pascuorum, B. hortorum, B. ruderatus) across an agricultural landscape centred on the Hillesden Estate, Buckinghamshire, UK. Locations were recorded in the field using a handheld GPS unit. Workers were non-lethally DNA sampled between June and August 2011, and genetic analysis used to confirm species and assign individuals to full-sib groups (colonies). Data were collected as part of a project led by the Centre for Ecology & Hydrology, funded under the Insect Pollinators Initiative.