Plantago is a large genus with a worldwide distribution characterized by three basic sets, x = 6 is the most common and is considered the ancestral set from which x = 5 and x = 4 derived following mechanisms of descending dysploidy. Advanced molecular cytogenetic methods are important for understanding evolutionary relationships but in Plantago their use has been rather limited. In this study the karyotypes of Plantago lanceolata L. (2n = 12), Plantago major L. (2n = 12) and Plantago coronopus L. (2n = 10) were characterized by using rDNA FISH, GISH, fluorochrome banding and chromosome morphology to obtain markers useful for evaluating their genomic affinity. The rDNA FISH mapping produced three distinct patterns but with evident similarities such as the chromosome position of 45S rDNA sites and the colocalization of 45S and 5S rDNA. Reciprocal GISH showed a low level of genome affinity between P. lanceolata and P. major and a total absence of homology between them and P. coronopus. This result supports the exclusion of the 2n = 12 species as possible ancestors of P. coronopus. This study confirms that a systematic use of in situ hybridization techniques, and in particular GISH, provide valid information for understanding the origin of species and can resolve many taxonomic and phylogenetic problematics.