Genetic structure and disturbance among populations of the Japanese perch Coreoperca kawamebari in Tokushima Prefecture were surveyed by mitochondrial DNA (mtDNA) direct sequence analysis. Two sample series were taken during different sampling periods from three river systems (Kuwano, Fukui, and Tsubaki River systems): (1) 1999, samples from river populations free of any officially sanctioned artificial introduction of seeds; and (2) 2011–14, samples from the Tsubaki River population previously subjected to artificial introduction of seeds from the Kuwano River population. In the 1999 samples, perch in the above three river systems all exhibited the Tokushima clade (comprising haplotypes Hap 1 and Hap 2). Hap 1 was shared by the Fukui and Tsubaki River systems, whereas Hap 2 was endemic to the Kuwano River system. A few cases of an additional haplotype (Hap 10), included in a different clade (Western Setouchi clade), were found in the Fukui River population. In the 2011–14 samples, Hap 2 of the Kuwano River endemic haplotype was shared with Tsubaki River populations, at rates from 25% to 56% among individuals of different year classes, suggesting mtDNA intrusion from the Kuwano River population. Additionally, all individuals in a tributary of the Kuwano River system (a recently discovered habitat of the species), exhibited a haplotype (Hap 11) belonging to the Western Setouchi clade. These findings suggest artificial intraspecific introductions among river populations, resulting in serious irreversible disturbance to the natural perch populations. Further detailed nuclear DNA surveillance should be conducted to clarify the ongoing status of the perch.