During the last decade, many scientists have developed microsatellite markers for genotyping and identify closely related genotypes. For citrus, such as for many other plant species, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. Recently, in a project associating CNS Genoscope, INRA, CIRAD (France) and IVIA (Spain), nearly 40 000 EST clones of different clementine organs have been sequenced, clustered and noted. Among 1500 unigenes, we have found microsatellites sequences constituted by units upper than one repeated nucleotide. More than 95% of these microsatellite sequences were di or tri-nucleotides with different repartition for each. If trinucleotide microsatellites can be encountered trough all part of EST sequences, dinucleotide microsatellites seem to be concentrated preferentially in the first 150 nucleotide 5¿ part. We have also assessed the polymorphism of these two kinds of sequences, by PCR amplification droved with boarding primers among thirteen Citrus species plus 3 other genera. From 50 analysed microsatellites, we have observed more than 90% of polymorphic loci. Furthermore, dinucleotide sequences were more variable than trinucleotide, probably related to their position that is more often not included in the open reading frame. These new markers located in gene sequences, offer new opportunities for genetic diversity studies in relation with known functions and facilities to map directly genes involved in the expression of identified characters. (Texte intégral)