Evidence for the molecular basis of a null allele of cinnamyl alcohol dehydrogenase (CAD) has been discovered in the loblolly pine (Pinus taeda L.) clone 7-56. The mutation is a two-base pair adenosine insertion located in exon 5 that causes a frame-shift which is predicted to result in premature termination of the protein. For routine detection of the mutation, a diagnostic assay was developed utilizing Template-directed Dye-terminator Incorporation and Fluorescence Polarization detection (FP-TDI). Loblolly pine is the most important commercial tree species in the USA, being harvested for pulp and solid wood products. Chemical pulping could be increased in efficiency by selecting for trees having a two-base pair adenosine insertion, by use of the rapid diagnostic assay developed in this study.