Expressed sequence tags-derived simple sequence repeats (EST-SSRs) are related to functional genes and can be useful in crop breeding. Parental lines selected in sweet potato (Ipomoea batatas (L.) Lam.) breeding including controlled hybridization and polycross may be originated from a limited number of genetic sources. We surveyed genetic variation of three breeding populations (hybrid, landrace, and polycross) of 89 cultivated sweet potato varieties derived from controlled hybridization (41 hybrids) and polycross (38 varieties) breeding population and landrace (10 varieties) maintained by various geographic regions and main agronomic traits of local farmers, using 23 EST-SSRs. Six wild sweet potato (I. trifida) samples were also included in the study. High genetic diversity in the three types of breeding populations was detected. High proportions of shared alleles leading to low levels of differentiation between breeding populations were discovered. The neighbor-joining tree and individual assignments showed intermingling of varieties among the three breeding populations. Additionally, genetic clustering showed little power of grouping varieties of different breeding populations into separate clades. These results indicate limits of sweet potato breeding. Nevertheless, ten alleles of nine loci showed variance contributed significantly to the delimitation of varieties in different breeding populations.