Recent studies utilizing transcriptome sequences generated by next-generation sequencing (NGS) technologies have demonstrated the ability to rapidly detect and characterize thousands of gene-based microsatellites from different plants. However, these simple sequence repeats (SSRs) were seldom used directly to test interspecific transferability in the populations of closely related species. Aspidistra Ker-Gawl. is a monocot genus with high species richness and diversity in flower structure, but its fresh floral materials are not easy to obtain. Until now, little is known about genetic background in the species of Aspidistra, quite apart from the fearful reduction of their natural habitats. In this study, the floral transcriptome of Aspidistra saxicola was obtained using NGS. Based on these data, a total of 5527 SSRs were identified in the unigenes. Among these SSRs, the proportions of di- and tri-nucleotide repeats were quite close (49.6% verse 46.8%), and the most tri-nucleotide repeats were AGG/CCT followed by AAG/CTT and AGC/GCT in A. saxicola, showing distinct differences with other angiosperm species. To assess genetic diversity in the species of Aspidistra, 48 SSR loci were tested in four available populations of A. elatior. The results revealed that more than a third of the loci were polymorphic. The majority of these primers could be amplified in 24 species representing the main clades of Aspidistra. The primer subsets from transcriptome data proved highly useful for detecting polymorphisms in the related species, supporting the finding that NGS is an efficient approach to molecular marker development at both intra- and interspecies levels, especially in endangered nonmodel species.