Anatomically preserved land plant fossils from the Lower Devonian Rhynie Chert contain conducting tissues with cells that range from dark-colored, elongated cells without secondary wall thickenings to tracheids similar to those of extant tracheophytes. A suite of tissue-specific microanalytical techniques was used to assess lignification in fossils of Aglaophyton, Rhynia, and Asteroxylon. Isotope ratio mass spectrometry provides millimeter-scale resolution of carbon isotopic abundances, whereas soft X-ray carbon (1s) spectromicroscopy provides micrometer-scale resolution of the preservation of organic molecular functionality. The isotopic and organic chemistry of Rhynie Chert plants indicates that the earliest vascular thickenings were probably unlignified and that cell wall lignification may have first appeared in the outer cortex. Only in more derived plants, it seems, was lignin deposited in conducting cells to produce the true tracheids seen today in vascular plants.