Molecular systematic studies and population genetics of Rhinogobius brunneus species complex were carried out by allozyme electrophoresis and mtDNA sequences analysis. The phylogenetic trees derived from these two data sets were indicated different topologies. Both allozyme and mtDNA data provided no evidence to support that the most species of R. brunneus species complex in Taiwan and the different color types in Ryukyu Islands were valid. The specimens from Ryukyu Islands form a monophyletic group within the species complex. The genetic distance of R. nagoyae formosanus (Rhinogobius sp. CB) between Taiwan and Ryukyu Islands was larger than that between different species and color types in the same area. Only the endemic R. rubromaculatus and R. maculafasciatus in Taiwan were valid species. Two hierarchical examinations of 27 populations using analysis of molecular variance (AMOVA) indicate high genetic differentiation among different islands (52.10%) and different populations (70.95%). The results support the hypothesis that the genetic relationship of R. brunneus species complex is weak contingency with color patterns and is otherwise possibly caused by geomorphological processes that occurred in the past. Therefore, the species nominated for the R. brunneus species complex should be reorganized. An additional color type discovered from the Linbian River in southern Taiwan is morphologically and genetically closely related to Rhinogobius rubromaculatus with little variation. Specimens from the Linbian River characterized by a smaller body size and egg size, are negatively correlated with thermal gradient. There is no fixed alleles between the new color type from Linbian River and R. rubromaculatus, with extremely lower genetic distance of 0.001 to 0.078, when compared to R. candidianus (0.523~0.687) and two other undetermined Rhinogobius species from the Ryukyu Islands (0.326~0.464). The complete sequences of the cytochrome b gene, tRNA genes, and the control region of mtDNA revealed substitution differences within R. rubromaculatus at 207 base pairs (bp) and among the three species (R. candidianus, Rhinogobius sp. BB, and Rhinogobius sp. YB) at 363 bp. The sequence diversity within the population of R. rubromaculatus ranged 0.000-0.004. Among local populations of R. rubromaculatus, the mean sequence distance diversities ranged 0.005-0.074, is far lower than that between R. rubromaculatus and other Rhinogobius species (0.094~0.113). The results from both genetic markers revealed the same significant differences between local populations of R. rubromaculatus (global FST = 0.404 for the allozyme and ΦST = 0.986 for the mitochondrial DNA data). The molecular trees constructed for R. rubromaculatus based on both allozyme and mtDNA data revealed a higher similarity between central and southern populations than to northern population. The phenotypic differences could have been produced by phenotypic plasticity. The genetic diversity within and among populations of the endemic spot-banded goby, Rhinogobius maculafasciatus, from three drainage basins in Taiwan was derived from a sequence length of 2124-2126 bp, including the complete cytochrome b gene, two tRNA genes, and the control region of mtDNA. Forty-one haplotypes obtained from sequence analysis of 60 specimens indicated two distinct clades in the samples from Kaoping River, southwestern Taiwan. The populations inhabiting the Lanyang River in northern Taiwan is allied to that in southwestern Taiwan, and both are probably originated from the east coast of Mainland China via Current Taiwan Striat by aide of the Coastal Current on along the western coast of Taiwan. A hierarchical examination of six populations of R. maculafasciatus from three drainage basins using analysis of molecular variance (AMOVA) indicates high genetic differentiation among populations within basins (68.37%). Most of the variation deduced from the population differentiations in Kaoping River. The results support the hypothesis that the current genetic structure of R. maculafasciatus is strongly affected by changes in drainage patterns due to geomorphological processes that occurred in the recent past.