Tomoplagia (Diptera: Tephritidae) are endophagous herbivores on flowerheads of Asteraceae, with varying levels of host specialization. We isolated, characterized, and tested the heterologous amplification for sets of microsatellites of three species from this genus associated with native hosts in the Espinhaço mountain range, Brazil. We used two protocols to build microsatellite-enriched libraries and characterized 12 polymorphic loci for each fruit-fly species. The samples of T. grandis, a monophagous species, presented a lower number of alleles than the samples of the oligophagous species T. incompleta and T. bicolor. The average observed heterozygosity did not vary among species and the high values of Polymorphism Information Content suggest that the characterized microsatellite loci are quite informative. The primers developed for each Tomoplagia species failed to amplify the microsatellite loci for the other two species, which indicates a low conservation of the flanking nucleotide sequences in the three evaluated species. The sets of polymorphic microsatellites characterized for these species of Tomoplagia may be used in future ecological and evolutionary studies that aim to detect patterns of diversity on a fine scale.