As a "living fossil" that is used to understand the evolutionary history of seed plants, Ginkgo biloba is a well-known multipurpose tree with edible seeds, medicinal properties, and ornamental value, but little is known about its genetic diversity. Microsatellite, or simple sequence repeat (SSR), markers have proven to be powerful tools for genetic studies of plants. In this study, we isolated 30 novel polymorphic microsatellite loci in G. biloba using 454 pyrosequencing. The characteristics of these loci were tested with 48 cultivars. The number of alleles (NA) per locus ranged from two to seven. The observed (HO) and expected (HE) heterozygosities ranged from 0.000 to 0.750 and from 0.021 to 0.792, with an average of 0.326 and 0.443, respectively. In terms of genetic diversity in the Ginkgo population, NA was 3.300, NE was 2.090, I was 0.782, HO was 0.326, and HE was 0.443. These polymorphic SSRs will be useful for the assessment of population genetic diversity and resource conservation of G. biloba.