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. Nested clade analysis identified two major clusters of the five haplotypes, one clade in the Yunnan region and the other in Hainan–Laos. The analysis indicated that restricted gene flow with isolation by distance and allopatric fragmentation were likely the major processes that shaped the spatial distribution of the haplotypes. The isolated distribution of clades implied the emergence of independent refugia of this species in each region during Quaternary glaciations. The Yunnan populations frequently contained an ancestral haplotype, and most of them harbored other descendent haplotypes. Based on the distribution pattern of haplotypes and the nested clade analysis results, the Yunnan region potentially had several refugia of this species during glacial periods, whereas the Hainan populations were probable new colonizations.