A rapid and reliable technique is needed for identifying butternut trees (Juglans cinerea) with resistance to butternut canker. We investigated the potential of a bark extract bioassay to detect levels of resistance to Ophiognomonia clavigignenti-juglandacearum (Oc-j), the causal agent of butternut canker. Both reagent grade naphthoquinones and crude bark extracts of Juglans species inhibited germination of Oc-j conidia. A disc diffusion bioassay was used to study the level of inhibition by these bark extracts and results indicated extensive variation within and between butternut and other species of Juglans tested. In many months over a 3 year period, bark from butternut trees selected for apparent disease resistance could be distinguished from that of unselected trees. Inhibition of conidia germination roughly correlated to the level of resistance observed in field inoculations of the trees. Quantification of the naphthoquinone compounds juglone and plumbagin in butternut bark was performed using ultra-high performance liquid chromatography mass spectrometry. While the concentrations of these two compounds varied by month and by individual tree, juglone levels correlated well with the bark extract bioassay in some months. These results suggest that juglone concentration may account in part for the observed range of inhibition observed in the bioassay and variation in canker resistance among selections of butternut field inoculated with Oc-j. The bark extract bioassay described in the following report may have potential use for selecting resistant butternut for conservation and restoration purposes.