Chemical analysis of the hydroalcoholic extracts from the aerial parts of the two accepted varieties of Ageratina espinosarum (A.Gray) R.M. King & H. Rob. (Asteraceae) resulted in the identification of ent-dihydrotucumanol (4), ent-dihydrotucumanoic acid (5), apigenin (19), acacetin (21), apigenin 7,4'-dimethyl ether (22), kaempferol (25), kaempferol 7,4'-dimethyl ether (26), and a mixture of 26 and kaempferol 3,7,4'-trimethyl ether (27) from A. espinosarum var. subintegrifolia; and taxifolin (7), naringenin (9), crysoeriol 4'-methyl ether (10), alysifolinone (11), sakuranetin (12), a mixture of sakuranetin 4'-methyl ether (13) and persicogenin (14), homoeriodictyol (15), and apigenin (19) from A. espinosarum var. espinosarum. Compounds 9, 12, 19 and 25 have been previously isolated of the aerial parts from this plant. Compound 19 was present in both varieties herein analyzed. Compounds 4, 5, 7, 10, 11, 13-15, 21, 22, 26, and 27 were isolated for the first time from A. espinosarum, despite this, the chemical content found in both varieties is consistent with that previously described. The flavanones, flavones and flavonols methoxy and dimethoxy derivatives are the chemophenetic markers of A. espinosarum. The structure of the known compounds was determined by spectroscopic and spectrometric techniques and their data compared with those previously reported. This is the first report of the isolation of compound 4, which was characterized as its 2,3,15-triacetyl (4A) and 2,15-diacetyl (4B) derivatives, whose spectroscopic and spectrometric data are described within.