In the present study we assessed silver-staining method and fluorescent detection assay using 9 SSRs and 85 peanut cultivars. The results showed fluorescent detection assay was more sensitive, accurate, and efficient than silver-staining method. Cluster analysis indicated fluorescent detection assay was able to separate 82 peanut cultivars into 9 clusters, while silver-staining method separated 74 cultivars into 8 clusters. The clusters from fluorescent detection assay were more closely related to the types, origins and pedigrees of the peanut cultivars. Genetic diversity analysis showed the peanut landraces had the highest genetic diversity index, followed by the Valencia type peanut cultivars, suggesting the landraces and Valencia type cultivars harboring a broad genetic variation.