We describe the first 134 polymorphic microsatellites loci developed for the fly Drosophila mediopunctata. A genomic DNA library double-enriched for dinucleotides was constructed and approximately 600 unambiguous microsatellites were identified. The DNA library consisted of 60% positive clones, short-sized dinucleotides (mean = 8.7 repeats) and 73% of AC/GT combinations. A total of 177 primer pairs were designed and thirteen strains were used to achieve optimal PCR amplification conditions and allelic variability of the isolated loci. The average number of alleles per locus was 4.6 and means of observed and expected heterozygosities were 0.23 and 0.62, respectively. The discriminating power average was 0.72. 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