The fluvial sculpin (Cottus pollux) is distributed in the Honmyo River located in Nagasaki Prefecture as the southernmost range. The suitable habitat for sculpins is restricted to about 1 km flow channel in the river. Fin samples from 1,301 sculpins were collected from 8 populations in each habitat in successive three year (2005-07). AFLP was applied to these populations. The genetic structure estimated by using Structure (Ver. 2.3.1) order of the body length exhibited two patterns, and a total of five distinct groups were found in each habitat population. The age analysis using the statolith revealed that these groups were consistent with the generations. In addition, the pattern disagreed between same generations in each sampled year, but did between same age classes. Furthermore, the five distinct groups were detected in another local populations in the southernmost range. The gonadosomatic index of over 1+ aged sculpins showed that some individuals remained to be immature in the end of the spawning season in the Honmyo River. From these results, we assumed that the five distinct groups result from not only the individual specificity, but from any temporal causes such as RNA transcripts. We also assumed that because the suitable low temperature seasons for maturity are too short for these sculpins in this southernmost range, they attain to maturity every other year.