Simple sequence repeats (SSRs) were isolated from a size-fractionated genomic DNA library of watermelon (Citrullus lanatus L. cv. New Hampshire Midget). Screening of the library with five oligonucleotide probes, including (GT)11, (AT)11, (CT)11, (GC)11, and (TAA)8, detected the occurrence of 96 positive colonies among ≈8000 recombinants. Automated DNA sequencing revealed the presence of SSRs. PCR primer pairs homologous to the regions flanking the SSR loci were synthesized commercially and used to screen 56 watermelon genotypes for the occurrence of SSR polymorphisms. Amplification products were separated using nondenaturing PAGE. Eighty percent of the primer pairs produced amplification products of the expected size and detected polymorphisms among the genotypes examined. The use of SSRs for watermelon germplasm characterization is discussed.