To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa (Layia hieracioides and Madia sativa), using phylogenetic inference and principal component analysis (PCA). Several groups supporting multiple accessions of the same species (of A. montana, A. longifolia, A. gracilis, and A. chamissonis) are congruent between the phylogenetic trees based on nrDNA and STL data. Sesquiterpene lactone profiles were found to be highly consistent within multiple samples of A. montana and A. longifolia respectively. Moreover, sesquiterpene lactone data support subspecies classifications of A. chamissonis and A. parryi, with additional support from DNA sequence data and cytometric data. Morphology, STL data (PCA), cytometric data and DNA sequence data suggest a hybrid origin of one accession (A. gracilis · longifolia). In A. gracilis, A. latifolia, and Layia hieracioides, previously not investigated for STLs, we found large amounts of xanthalongin derivatives. This is the first time STLs have been reported from subtribe Madiinae.