(E)-Resveratrol is a naturally occurring polyphenolic compound in plants with a variety of widely studied health benefits. The bark of Northern American, Canadian, and Northern European conifer species, which is an underutilized by-product generated by forest industries, is a source of (E)-resveratrol, providing a potential value-added product for these industries. Bark may serve as a good alternative to the invasive plant Japanese knotweed (Polygonum cuspidatum), which currently is the leading commercial source of (E)-resveratrol. This work describes a method to extract and purify (E)-resveratrol from conifer bark with high yield and high purity and investigates the relationship between the amount of (E)-resveratrol and the total phenolic contents in the bark of common conifer species. In this work, barks of four conifer species were extracted and the total phenolic contents were determined by Folin−Cicoalteu’s assay. The (E)-resveratrol content was determined by HPLC-MS. A purification method that utilizes solvent extraction and column chromatography was developed to isolate (E)-resveratrol in high yield from black spruce (Picea mariana) bark. The quantitative analysis of bark samples suggests the presence of (E)-resveratrol in black spruce (Picea mariana) and Norway spruce (Picea abies), in comparable amounts to Japanese knotweed. Based on HPLC-MS and HPLC-UV analyses, the purification method isolates the compound with a yield of 84% and purity of 99%. Hence, our method extracts and isolates (E)-resveratrol from conifer bark in high purity and high yield. The results do not support any correlation between the total phenolic content and the amount of (E)-resveratrol.