Alpinia oxyphylla Miquel is an endangered but economically valuable species. Given that it is one of the four most important medicinal plants in southern China, surprisingly few studies have characterized the species at the molecular level. In this study, 163 accessions were collected from seven natural populations of A. oxyphylla, along with one outgroup species, to investigate the genetic diversity of the species using intersimple sequence repeat (ISSR) analysis. A total of 365 polymorphic markers were detected from the general population when it was analyzed using 20 ISSR primers. Nei's genetic diversity and Shannon's information index were 0.2129 and 0.3373, respectively, indicating considerable genetic diversity at the species level. Less genetic differentiation took place among populations, with evidence of coefficient of gene differentiation 0.338 and gene flow 0.907, suggesting that there was more genetic variation within populations than between populations. An unweighted pair group method with arithmetic averaging dendrogram divided all accessions into eight groups that coincided almost completely with their geographical origin, with the exception of just a few crosses. This suggests that the geographic distribution of accessions coincided closely with the distribution of genetic diversity within A. oxyphylla, indicating weak gene flow. These results may provide valuable guidance regarding the conservation and genetic improvement of the species.