Phalaris aquatica L. (phalaris) is a cool-season perennial grass originating from the Mediterranean Basin, north-west Africa and Middle Eastern regions that is used for livestock agriculture mainly in temperate areas with dry summers. It has been the subject of breeding programs in Australia, South America, New Zealand and the USA. Increased knowledge of relationships between wild and cultivated germplasm through use of molecular markers has the potential to facilitate future breeding gains. For this purpose, we conducted an analysis of P. aquatica by using 3905 polymorphic DArTseq SNP markers. Genetic diversity as measured by expected heterozygosity was similar for wild (HE = 0.14; n = 57) and cultivated (HE = 0.13; n = 37) accessions. Diversity in wild germplasm was generally continuous in nature, largely related to geographical location, with a division at the broadest scale into eastern and western clades, with more admixture in the western than the eastern clade. Structure analysis of wild germplasm indicated six subpopulations consistent with country of origin, with some admixture among subpopulations likely resulting from natural and human influences. There were nine subpopulations among wild and cultivated accessions combined. This population structure should be considered if genomic selection is applied in P. aquatica. Analysis of molecular variance indicated that 71% of the genetic variation occurred within subpopulations and 29% among subpopulations. Genetic distances were low among cultivated germplasm from most countries except the USA, which was more distinct. Evaluation of material from the US pool by breeding programs in other countries, and additional material from the less utilised eastern clade, may be worthwhile.