A molecular cytogenetic study has been performed in three species of the genus Artemisia, complementing previous works on two subgenera that had been scarcely studied from this standpoint, Artemisia (A. chamaemelifolia, A. vulgaris) and Absinthium (A. absinthium). Chromomycin A 3 and 4',6-diamidino-2-phenylindole (DAPI) banding have been carried out, as well as fluorescent in situ hybridization (FISH) of 5S and 18S-5.8S-26S ribosomal DNA. Morphometrical data of karyotype characters were calculated and idiograms with the position of the AT-and GC-rich regions as well as rDNA loci were constructed. Colocalization of most of these regions has been observed, confirming previous findings in this genus. Both ribosomal DNA appear always colocalized, which is a distinct feature with respect to most angiosperms surveyed. Regarding the differential characteristics of each species, a symmetrical karyotype has been found in the species studied. Artemisia absinthium shows long chromosomes and absence of centromeric banding signals that, conversely, are absent in A. vulgaris and A. chamaemelifolia. The last species also presents Bchromosomes in which ribosomal DNA and heterochromatin have been detected. Despite these differences, karyotype morphology and signal pattern of the three species are quite coincidental. This might reflect a close phylogenetic relationship between both subgenera, which is consistent with the available molecular phylogenies presenting species of the subgenera Artemisia and Absinthium intermixed.