Sequences of the internal transcribed spacers (ITSs) of 18S-26S nuclear ribosomal DNA were used to resolve phylogenetic relationships and chromosomal evolution among 14 species of the genus Hypochaeris (Asteraceae). Parsimony analysis was performed for phylogenetic reconstruction, and sequence divergence between species was estimated. Pairwise sequence divergence within Hypochaeris genus ranged from 0% to 25.68% in ITS1 and from 0% to 17.08% in ITS2. A highly resolved strict-consensus tree was obtained that showed the phylogenetically useful information of ITS sequences within the genus Hypochaeris. Four clades could be well distinguished, one of them formed by the single species H. robertia, which appeared to be the most related to the ancestral species of the genus. The results agree with taxonomic classification based on morphological data, and the tree obtained, when indels are coded as missing data, aggregates the species having the same chromosome number, except in one clade. According to the ITS phylogenetic tree, the chromosomal evolution within the genus Hypochaeris conflicts with the previous hypothesis and suggests that karyotype evolution in Hypochaeris was accompanied with both decreasing and increasing dysploidy, probably with several chromosomal rearrangements, and from an ancestral basic chromosome number of 4 or 5.