Lichens are specialized complex ecosystems formed by algae/fungi living in interspecific symbiosis, which produce secondary metabolites that function as a defense against pathogens/competitors and/or external abiotic factors acting as antibiotics, antifeedants, UV protectants, etc., but also as a tool for taxonomy of lichen-forming fungi. However, the lichen substance content of crustose lichens of the genus Graphis from Colombian tropical dry forests has been poorly explored. In this work, the chemical composition of total extracts from three lichens belonging to the genus Graphis collected in the tropical dry forests of northern Colombia was determined by TLC/HPLC–DAD/MS (ESI); and from one of them, two depsidones were isolated/structurally characterized (LC-HRMS, 1H-13C (DEPT135, HSQC)-NMR). Consequently, the main findings were as follows: (i) stictic (1)/constictic (2), norstictic (5), and protocetraric (13) acids were the representative depsidones for G. dendrogramma (Tubará), G. dendrogramma (Piojó), and G. supracola, respectively; and (ii) the isolated depsidones from G. dendrogramma (Tubará) were stictic (−)-(1) and constictic (+)-(2) acids. These results provided key insights into the chemical diversity and taxonomic biomarkers of these species, and therefore, it could be concluded that (i) there was chemical variability between G. dendrogramma collected in different places, although all contained stictic acid–derived depsidones; (ii) for the first time, extrolites other than protocetraric acid (13) were identified for G. supracola, as well as norstictic acid (5) as major lichen substance for G. dendrogramma; (iii) the main chemical biomarker [stictic acid (1)] reported in the scientific literature for one of G. dendrogramma sample (Tubará) was confirmed (by LC–MS and NMR analysis); and (iv) for the first time, the specific optical rotation related to the chirality of the two isolated depsidones was determined [(−)-(1), (+)-(2)]. Keywords: Graphis dendrogramma · Graphis supracola · Stictic acid–derived depsidones · Corticolous lichens · Tropical dry forest.