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Prosopis chilensis and Prosopis flexuosa (Fabaceae) are closely related hardwood arboreal species that are widely distributed in the arid regions of Argentina. The development of highly polymorphic markers, such as microsatellites, is desirable for genetic studies of these species. Here, we present the development and characterization of six polymorphic microsatellite markers in P. chilensis and P. flexuosa. These markers showed a polymorphism information content between 0.14 and 0.85 and the number of alleles varied from two to 13 considering both species. All markers revealed a broad cross-species affinity when tested in seven other Prosopis species. All primers amplified in at least five species.