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The <i>Elaeagnus</i> L. species are trees and bushes that mainly grow in temperate zones of Western Europe; Minor, Central, and Southeast Asia; the Far East; and North America. Some species are used as fruit or ornamental plants and have economic value. Problems with the identification of species in the <i>Elaeagnus</i> genus by molecular genetical methods arise in the study of populations, systematics, breeding, and other areas of plant science and practice. Recently, the polymorphism of 5S ribosomal DNA non-transcribed spacers (5S rDNA NTSs) in Elaeagnaceae Adans. has been described. The results were used in our study as a basis for development of new species-specific molecular markers for some members of the <i>Elaeagnus</i> genus. The author’s method was applied for finding regions that were potentially applicable for species-specific primer design. As a result, some species-specific molecular markers were developed for <i>Elaeagnus angustifolia</i> L., <i>E. commutata</i> Bernh., <i>E</i><i>. pungens</i> Thunb., and <i>E. multiflora</i> Thunb. These markers were tested in a range of samples and showed the presence of amplified fragments in lanes of the marked species only. Samples of other species showed no amplifications. Thus, the developed markers may be useful for the species identification of the studied <i>Elaeagnus</i> plants in botanical, dendrological, and genetic research (especially in a leafless period of year), as well as in breeding and hybridization experiments.