Ramie (Boehmeria nivea L. Gaud) is one of the most important natural fibre crops. For enhanced crop development, it is necessary to understand its population structure and genetic relationships. In this study, we assessed the genetic diversity and population structure of 134 ramie accessions (with three plants per accession) from 12 regions by using 36 simple sequence repeat markers. The 36 microsatellite primers revealed 149 alleles in 134 ramie populations, with an average of 4.14 alleles per locus. The structure analysis divided the 134 ramie accessions into three groups (I, II and III), and into further six subgroups (a, b, c, d, e and f). In Subgroup b, 13 accessions were from Guizhou Province, 9 accessions were from Sichuan Province and the remaining 20 accessions were from Chongqing (4), Hunan (8), Guangxi (4), Jiangxi (2), Yunan (1) and Taiwan (1). In Subgroup d, 22 accessions were from Guizhou Province and the remaining 17 accessions were from Chongqing (6), Sichuan (5) and Yunnan (6). It can be inferred that the genetic background of these ramie accessions did not always correlate with their geographical regions. Similar results were found in Subgroups a and f. The pair-wise genetic similarity coefficients between the 134 accessions ranged from 0.390 to 0.939, which suggested that there was abundant genetic diversity in the ramie accessions. These markers have provided important information about the genetic structure of ramie, which can contribute to future breeding and improvement programmes for these resources.