A freshwater minnow (Zacco platypus) is a freshwater fish widely distributed in South Korea. The study was conducted in order to confirm genetic variation with regards to water system via sequence of the mitochondrial 16S rRNA. The freshwater minnows used in the study was collected from 10 sites in four watersheds systems of the Hangang, Geumgang, Yeongsan Seomjingang and Nakdonggang. Sequence analysis of the 16S rRNA using PCR revealed that sequence length is 624bp. According to Bayesian Analysis, unique sequence was determined in two freshwater minnows, Tamjingang A and Yeongsangang A, of the Yeongsan Seomjingang water system. There was no sequence variation which was specific to the individual Hangang, Geumgang and Nakdonggang water systems. The 16S rRNA sequence of the Korean freshwater minnow as recorded by the NCBI showed 94% sequence similarity to the Japanese minnow (Zacco sieboldii). Study result was not able to clearly confirm sequence variation regards to geographical classification using the 16S rRNA alone in this species. Therefore, there is a need for analysis with regards to geological differences using Cytb or other nuclear sequence.