Previous microsatellite analysis showed that two subpopulations of perch (Perca fluviatilis L) exist in Lake Constance. This raises questions of whether (i). Lake Constance was colonized by two populations that diverged in allopatry, or (ii). the two subpopulations diverged in sympatry. Sequence analysis of a 365 bp mtDNA fragment (5'-end of the D-loop) of perch from Lake Constance and adjacent waters revealed 10 haplotypes. We suggest colonization via the Danube river, based on the frequency and dispersion of haplotypes, and knowledge of the lake's palaeohydrological development. Pairwise FST-values using mitochondrial DNA sequences showed no significant population subdivision. Our study provides strong evidence that subpopulations of perch in Lake Constance have diverged in sympatry.