The invasive cyanobacterium Cylindrospermopsis raciborskii is increasingly spreading to temperate freshwater habitats world wide and is of major concern due to its ability to produce potent toxins. It is therefore important to understand the mechanisms behind the dispersal of this species. Different hypotheses have been proposed to explain the phylogeography and mechanisms underlying the recent expansion of C. raciborskii into temperate latitudes, but there is still no conclusive evidence whether the obvious ecological success of C. raciborskii is due to selection mechanisms, physiological tolerance, climatic change or radiation after the last ice age. In this study, new isolates of C. raciborskii from Europe and Africa were genetically characterized by sequencing the ITS1, PC-IGS, nifH and rpoC1 genes and compared to corresponding sequences of C. raciborskii available in GenBank in order to test different phylogeographical hypotheses. The strains were also morphologically examined and screened for production of the hepatotoxic cylindrospermopsin (CYN). We clearly demonstrate that there are phylogenetic, morphological and toxicological differences between the isolated strains. The phylogenetic analyses revealed a clustering of the strains due to geographic origin. The ITS1 and nifH genes separated into American, European and Australian-African groups, whereas the PC-IGS and rpoC1 separated into American and European/Australian/African groups. Our findings do not strongly support any of the existing hypotheses on the phylogeography of C. raciborskii, and most likely a combination of these hypotheses is the best approach to understand the evolution and dispersal of this species.