Alerce (Fitzroya cupressoides (Mol.) Johnst.) and Guaitecas cypress (Pilgerodendron uviferum (Don) Florin) are two of the three closely-related species of conifers in the Cupressaceae that are endemic to southern Chile and Argentina. Both are listed in Appendix I of the Convention on International Trade in Endangered Species of Fauna and Flora (CITES). The presence or absence of nodular (conspicuously pitted) end walls in the parenchyma cells provide good diagnostic characters to separate the two species wood anatomically, but the latter is sometimes difficult to distinguish. Therefore, a collaborative project was designed to study the chemical-molecular expression of these species by analyzing the heartwood using DART TOFMS (Direct Analysis in Real-Time (DART) Time-of-Flight Mass Spectrometry (TOFMS). This study compares the anatomical features of heartwood for both species and demonstrates that anatomy in conjunction with chemistry can separate them. DART TOFMS analysis combined with PCA was able to unequivocally determine taxonomic source with a statistical certainty of 99%. The mass spectra results obtained from heartwood demonstrated that identification is feasible after a few seconds, using a very small sample. DART TOFMS is a robust tool for reliable species identification and is useful to identify the taxonomic source of finished products or timber that are suspected of being illegally harvested.