Localized dispersal and mating may genetically structure plant populations, resulting in matings among related individuals. This biparental inbreeding has significant consequences for the evolution of mating systems, yet is difficult to estimate in natural populations. We estimated biparental inbreeding in two populations of the largely self-fertilizing plant Aquilegia canadensis using standard inference as well as a novel experiment comparing apparent selfing between plants that were randomly relocated within populations to experimental control plants. Using two allozyme markers, biparental inbreeding (b) inferred from the difference between single-locus and multilocus estimates of selfing (b = s_s − s_m) was low. Less than 3% of matings involved close relatives (mean b = 0.029). In contrast, randomly relocating plants greatly reduced apparent selfing (mean s_s = 0.674) compared to control plants that had been dug up and replanted in their original locations (s_s = 0.953, P = 0.002). Based on this difference in s_s, we estimated that approximately 30% of all matings involved close relatives (mean b = 0.279, 95% CL = 0.072–0.428). Inference from s_s − s_m underestimated b in these populations by more than an order of magnitude. Biparental inbreeding is thought to influence the evolution of self-fertilization primarily through reducing the genetic cost of outcrossing. This is unlikely to be of much significance in A. canadensis because inbreeding depression (a major cost of selfing) is much stronger than the cost of outcrossing. However, biparental inbreeding combined with strong inbreeding depression may influence selection on dispersal.