The genetic structure of four populations of Pararasbora moltrechti, an endemic species of the Cyprinidae in Taiwan, was investigated based on the genetic variation of mtDNA Cyt b gene and five microsatellite loci. High haplotype diversity (h = 0.92) but low nucleotide diversity (0.004) in mtDNA was detected in this endangered species. In total, 33 haplotypes and four clusters were identified in its mtDNA. Nevertheless, low correspondence was found between geographical division and mtDNA clusters. In contrast, Bayesian cluster analysis of the microsatellite data identified four genetic groups and revealed highly structured populations. Significantly negative Tajima's D statistics and mismatch distribution analyses suggest that P. moltrechti populations may have experienced a demographic expansion. In light of the results of a nested clade analysis of mtDNA haplotypes, we conclude that recent population fluctuations and restricted gene flow played major roles in shaping the spatial genetic structure of P. moltrechti populations.