Understanding genetic diversity and population structure is essential for the conservation and utilization of germplasm. Ginkgo biloba L. is a medicinal, edible and ornamental tree species. Detailed knowledge of genetic variability and diversity in different Ginkgo germplasm resources is still scarce. In this study, a total of 173,160 Expressed Sequence Tag Simple Sequences Repeat (EST-SSR) loci were derived from 43,073 Unigenes of the Ginkgo genome. A total of 43,731 pairs of specific primers were designed for the EST-SSR loci, with sequence lengths >20 bp, and 100 of the SSR primers were randomly selected. Among these, 20 EST-SSR markers were verified and used to assess the genetic diversity of 101 Ginkgo individuals collected from different regions. The average values for Shannon’s diversity index (I, 0.993), expected heterozygosity (0.566) and Nei’s genetic diversity index (H, 0.563) indicate a high level of genetic diversity of Ginkgo populations. Based on the EST-SSR markers, a core collection of Ginkgo germplasm comprising 27 genetic resources was constructed. The retention rates of the number of resources, the number of alleles, the number of effective alleles, the I index, the H index and the percentage of polymorphic loci of the constructed core collection are 26.73%, 95.29%, 103.43%, 102.25%, 102.91% and 100.00%, respectively. The molecular markers developed in this study are an effective tool for Ginkgo genetic diversity analysis and will facilitate the future breeding of this species.