Geranium macrorrhizum L. is a herbaceous species native to southern Europe, and introduced in central Europe and North America. It is widely distributed also in Italy, up to Campania region as the southern limit. In this study, molecular and cytogenetic analyses were carried out on 22 wild plants, collected in central and southern Italy, compared to five cultivated plants, with the main purpose to identify those living near the Marmore waterfalls in central Italy, recently described as the new species G. lucarinii. Four barcoding markers (rbcL, matK, trnH-psbA intergenic spacer, and internal transcribed spacer region, ITS) were sequenced and their variability among the plants was evaluated. Chromosome numbers were determined and 45S rDNA was physically mapped by fluorescence in situ hybridization (FISH) in both wild and cultivated plants. Moreover, genomic affinity between wild and cultivated plants was evaluated by genomic in situ hybridization (GISH). Our results confirmed that all the plants, including Marmore population, belong to G. macrorrhizum. Barcoding analyses showed a close similarity among the wild plants, as well as a differentiation, although not significant, between the wild plants on one hand and the cultivated plants on the other. Integrated studies focusing on morphological, genetic and ecological characterization of a larger number of wild populations will allow for knowing the extent of the variability within the species.