The pawpaw [Asimina triloba (L.) Dunal.] is a tree fruit native to many areas of the southeastern and mid-western United States. Kentucky State University (KSU) is designated as a satellite repository for Asimina for the U.S. Department of Agriculture (USDA), National Plant Germplasm System (NPGS). An assessment of the level of genetic diversity in cultivated pawpaw would assist in development of the future germplasm repository collection strategies for cultivar improvement. The objectives of this study were to identify intersimple sequence repeat (ISSR) markers that segregate in a simple Mendelian fashion and to use these markers to assess genetic diversity in 19 pawpaw cultivars. Leaf samples from the 34 progeny of controlled crosses (1-7-1 × 2-54 and reciprocal) and the parents were collected, DNA was extracted, and subjected to the ISSR methodology using the University of British Columbia microsatellite primer set #9. Seven primers yielded 11 Mendelian markers with either a 3:1 or 1:1 ratio that was confirmed by chi-square analysis. Analysis of genetic diversity using 10 of the ISSR markers from 19 pawpaw cultivars revealed a moderate to high level of genetic diversity, with a percent polymorphic loci P = 80 and an expected heterozygosity He = 0.358. These diversity values are higher than those reported for cultivated pawpaw using isozyme or randomly amplified polymorphic DNA (RAPD) markers, indicating that the ISSR marker methodolgy has a higher level of discrimination in evaluating genetic diversity in pawpaw and/or pawpaw has greater levels of genetic diversity than previously found.