Sequence analyses of the non-coding, control region (CR) and coding region of the ND4-tRNA^Ser genes in the mitochondrial DNA (mtDNA) were conducted for populations of the ayu Plecoglossus altivelis altivelis and the Ryukyu-ayu P. a. ryukyuensis. The level of genetic differentiation between the two subspecies evaluated from the CR data was substantially low, when comparing with that estimated from ND4-tRNA^Ser gene region data, as well as those from nuclear genome data sets. By contrast, the differentiation between subspecies in the ND4-tRNA^Ser gene region was substantial, being consistent with the results from the previous nuclear genome analyses. Results of UPGMA and minimum spanning network analyses also implied the unexpected ceiling of genetic differentiation in the CR. These results suggest that the CR does not reflect accurately the level of overall genetic differentiation between the populations of the ayu, but other coding regions of the mtDNA do reflect it so that the mtDNA on the whole may function as a rich source of useful markers for genetic assessment of populations of this species.