Drosophila mediopunctata, a Neotropical fly that belongs to the tripunctata group, was chosen by some Brazilian researchers as a model in evolutionary studies. Several approaches, such as the analysis of natural variation and the influence of chromosome inversions in population dynamics, are traditionally used, but molecular markers are still not available for the species. Microsatellites are celebrated markers and the chosen tool for the exploration of various organisms due to their ease of use. This study intended to develop, characterize and map microsatellites for D. mediopunctata so that initiatives concerning morphological variation, identification of genomic regions linked to interesting phenotypes, population genetics, etc can be carried out in the light of molecular data. An enriched DNA library revealed 600 microsatellites from which 134 loci were developed. The microsatellites are small in length and mainly composed of AC/GT dinucleotides. Heterologous amplification with the D. mediopunctata microsatellites in other 30 Drosophila species was done with 50% success. A clustering analysis carried out with binary data obtained from the tripunctata species recovered already known phylogenetic relationships. A linkage map was constructed with recombination data of 49 markers and is 450 cM in length. The 5 major species chromosomes were identified on the basis of comparisons with the D. melanogaster genome and Müller elements. This strategy also confirmed the great synteny predicted for the genus Drosophila. It was not observed agreement in loci order between both species. A genomic region associated with the number of abdominal spots was mapped to D. mediopunctata chromosome II.