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The Atlantic Forest is one of the most threatened biomes in the world. Here, we use a common set of microsatellite markers to assess the genetic diversity and population structure of three species from the genus Psidium (P. guajava, P. macahense, and P. guineense), located in a disturbed environment of the Atlantic Forest, the restinga, in Espírito Santo, Brazil. Psidium guajava populations presented the highest number of alleles (95) followed by P. guineense (81) and P. macahense (68). The genetic variability was high (P. guajava = 0.71; P. guineense = 0.74; P. macahense = 0.63), with greater variation within populations (72 to 84%) than among populations (15 to 27%), reflecting elevated values of genetic differentiation (P. guajava, F ST: 0.15; P. macahense, Ø ST : 0.27; P. guineense, Ø ST : 0.21). The populations were clustered into two main groups and considered moderately structured. This is the first report of genetic studies and evidence of polyploidy to P. macahense. Our results may provide information that can be used in management and conservation strategies, to preserve the diversity of Psidium populations., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)