Microsatellite markers (SSR) have broad utility in genetic studies due to a high rate of polymorphisms, a codominant nature and multiallelism. EST–SSRs are markers derived from the expressed sequences of a genome and represent transcribed genes. Despite the importance of the genus Coffea, only a small number of EST–SSR markers are currently available. Thus, this study was designed to mine and develop a new set of EST–SSR markers from the Brazilian Coffee Genome Project. We investigated 130,792 expressed sequence tags (ESTs), from which 24,031 DNA sequences with microsatellites were identified. After stability and amplification testing, 101 new EST–SSR markers were developed and analyzed in different coffee species. The average rate of transferability was 88 %, showing that these markers are useful in genetic studies across the genus Coffea. Polymorphism levels and the degree of diversity were consistent with the evolutionary history of the species. All coffee genotypes were discriminated, even the C. arabica genotypes that have known narrow genetic basis. It was also possible to locate 14 EST–SSRs into different linkage groups of the C. arabica genetic map, which demonstrate that these markers can be useful in QTL mapping studies and in molecular-assisted selection.