Intraspecific macromolecule variation in stemwood, knotwood, and branchwood was studied using analytical pyrolysis with the intention of introducing a rapid working method to assess the variance in lignin content using analytical pyrolysis and highlight variability markers. The study was performed on Picea abies, Abies alba, and Pseudotsuga menziesii. Lignin determined via analytical pyrolysis–GC/MS (Py-lignin) can be used to identify variations in lignin content, compared to using classical Klason lignin values as a reference method for lignin determination, which requires a correction factor. Principal component analysis (PCA) was performed to identify biopolymer pyrolysis product markers for different species, tissues, or heights that could help highlight structural differences. Douglas fir was differentiated from spruce and silver fir in the levoglucosan amount. Guaiacol was more present in spruce wood, and creosol was more present in Douglas fir. Knotwood was structurally close to stemwood in spruce and silver fir, but there was a clear transition between stemwood and branchwood tissue in Douglas fir. Knotwood was differentiated by higher furan compounds. Branchwood was clearly separate from stemwood and knotwood and presented the same markers as compression wood in the form of phenylpropanoid lignins (H-lignin) as well as isoeugenol and vinyl guaiacol, the two most produced lignin pyrolysis products.