Microsatellite-containing sequences were isolated from an enriched genomic library of Carica papaya L. A total of 507 clones were evaluated with radioactive probes containing the motifs (GA)15 and (GT)15 and 147 positive clones were isolated and sequenced. The OLIGO 4.06 software was used to define 76 primer pairs. These primers were evaluated on three genotypes of C papaya and seven species of the genus Vasconcellea. Overall, 69 primer pairs worked in C papaya and 33 in Vasconcellea. The 35 pairs producing the best banding profiles were characterized on a sample of 29 individuals of C. papaya from the Caribbean region and 11 individuals of Vasconcellea from the Andes of Colombia and Ecuador. Twenty-six primer pairs gave polymorphism, of which 24 within C. papaya and only four within the seven Vasconcellea species, confirming the divergence between both genera. The number of alleles was 99, with a mean of 4 and an expected heterozygosity (He) of 0.56 per locus in C papaya. and 18, i.e. 4.5 per locus, in Vasconcellea, in the homozygous state. Microsatellite markers revealed more polymorphism in papaya than AFLP or RAPD.