Mitochondrial DNA (mtDNA) of Japanese Longfin Smelt Spirinchus lanceolatus among four river populations were examined to clarify their genetic feature and population structure. Restriction fragment length polymorphism (RFLP) analysis using 9 kinds of 6-base recognized restriction endonucleases (Apa I, Ava I, BamH I, Bgl II, BstE II, EcoR I, Pst I, Pvu II, Xba I) revealed considerable variaton. We found 17 haplotypes in 91 specimens. Restriction fragment patterns were polymorphic for 7 enzymes examined. Nucleotide divergence between haplotypes ranged from 0.27 to 2.33%. Nucleotide divergence between populations ranged from 0.37 to 0.54%. A dendrogram constructed by UPGMA among 17 haplotypes showed no clustering groups of localities. Nucleotide diversity and nucleon diversity of each locality ranged from 0.0028 to 0.0052, and from 0.7758 to 0.9074, respectively. Distinguishable two stocks by morphological analysis showed significant difference in their haplotype freqencies.