Genetic diversity of Coffea arabica cultivars was estimated using amplified fragment length polymorphism (AFLP) markers. Sixty one Coffea accessions composed of six arabica cultivars, including Typica, Bourbon, Catimor, Catuai, Caturra and Mokka Hybrid, plus two diploid Coffea species, were analyzed with six EcoRI- MseI primer combinations. A total of 274 informative AFLP markers were generated and scored as binary data. These data were analyzed using cluster methods in the software package NTSYSpc. The differences among cultivars at the DNA level were small, with an average genetic similarity of 0.933. Most accessions within a cultivar formed a cluster, although deviant samples occurred in five of the six cultivars examined due to residual heterozygosity from ancestral materials. Among the six cultivars fingerprinted, the highest level of genetic diversity was found within the cultivar Catimor, with an average genetic similarity of 0.880. The lowest level was found within Caturra accessions, with an average genetic similarity of 0.993. Diversity between C. arabica and two other Coffea species, Coffea canephora and Coffea liberica, was also estimated with average genetic similarities of 0.540 and 0.413, respectively, suggesting that C. canephora is more closely related to C. arabica than is C. liberica. The genetic variation among arabica cultivars was similar to the variation within cultivars, and no cultivar-specific DNA marker was detected. Although arabica cultivars appear to have a narrow genetic base, our results show that sufficient polymorphism can be found among some arabica cultivars with a genetic similarity as low as 0.767 for genetic/QTL mapping and breeding. The assessment of genetic diversity among arabica cultivars provided the necessary information to estimate the potential for using marker-assisted breeding for coffee improvement.