In this study, we analyzed the genetic structure and phylogeography of Sphaeropteris brunoniana from China and Laos. Combining cpDNA trnL-trnF and atpB-rbcL sequence variations, five haplotypes were identified from the 10 investigated populations. Moderate haplotype diversity (h= 0.66580) and low nucleotide diversity (π= 0.23 × 10−3) were detected. The S. brunoniana in Yunnan region had much higher genetic diversity (h= 0.60195, π= 0.35 × 10−3) than that of Hainan-Laos (h= 0.00000, π= 0.00). A high level of genetic differentiation (94.74%) between the two regions was revealed by amova