Technical Abstract: Cucumis metuliferus E. Mey. ex Naud (African horned cucumber, AHC) is native to Africa and is a relative of cultivated cucumber (C. sativus L.) and melon (C. melo L.). AHC is being promoted as a specialty fruit crop in a number of countries. In addition, several traits in C. metuliferus such as high level resistance to the several nematode species and papaya ring spot virus, sequential and concentrated fruiting are very attractive for improving cucumber or other cucurbit crops. However, attempts in the past to cross AHC with either cucumber or melon were unsuccessful. In the present study, genetic diversity among the USDA C. metuliferus collection was evaluated with microsatellite (simple sequence repeat, SSR) markers derived from cucumber genomic DNA sequences. Of the 564 cucumber SSRs tested, 51.8% were able to amplify PCR products from C. metuliferus genomic DNA templates suggesting high degree DNA sequence homology between the two species. Forty two cross-species transferable SSRs were selected to investigate genetic variability among 35 accessions from USDA collection and 1 commercial source C. metuliferus selections. Genetic diversity among these C. metuliferus accessions was low. Of the 42 markers tested, 13 were monomorphic among the 36 accessions, and each marker, on average, was able to detected only 3.4 alleles. Clustering analysis support a positive relationship of genetic distance and geographic distance among these accessions. The possible reasons of the low genetic diversity among C. metuliferus accessions and the implications of this finding with regard to use of C. metuliferus resources for cucurbit improvement were discussed.