An EST-based PCR-RFLP method was employed to gain insight into genome organization in eight allopolyploid Coffea arabica cultivars and seven diploid coffee species. The PCR-amplified products at 19 EST loci were digested with 46 different restriction enzymes and size fractioned in agarose gels. Most often, the sum of the fragments length was double or more than the PCR product. In arabica, this condition could be explained by assuming the presence of duplicated loci in paralogous chromosomes and this was supported by considerable evidence of multiple loci SSR amplification. Based on the RFLP analysis, 12 EST loci were polymorphic. The level of polymorphism was higher in different species compared to the arabica varieties. Sequencing of the amplified products revealed a SNP frequency of 0.021 among diploid species and of 0.007 among arabica varieties. We propose that the involvement of two genomes in C. arabica maintains a residual level of heterozygosity in the form of paralogous chromosomes, while the self-fertilization in this species tends to drive of homozygosity. The heterozygosity of paralogous chromosomes in arabica creates valuable polymorphism essential for species diversity and survival in various ecological niches, while self-fertility tends to preserve in homozygosity many genes of functional significance.