The distribution of populations of Greater Rhea (Rhea americana) are fragmented, probably owing to the conversion of grasslands to croplands. This study analyses the genetic structure of populations of Greater Rhea in areas with different degrees of anthropogenic alteration. Molecular variation was evaluated using Inter Simple Sequence Repeats (ISSR) markers in five wild populations that inhabit an agroecosystem and an area of semi-natural grassland. The populations were polymorphic for 23.33% of loci, with an average genetic diversity of 0.0822, and the populations of the two regions showed similar levels of variability. This low level of polymorphism may have be a result of historical bottlenecks and the long generation time of this species. The analysis of molecular variance showed highly significant differences among populations and non-significant differences between regions. The effects of habitat changes might not be the most important factor determining the genetic differentiation among populations currently. However, as modification of landscape structure of the Pampas grasslands could be more severe in the future, conservation of the Greater Rhea requires careful land-use planning.