A previous investigation demonstrated the existence of extensive allele frequency diversity within an area of northern Italy crossed by a linguistic (dialect) boundary and by the Po River, either of them or both presumably constraining gene flow. We obtained hair samples from 45 school pupils from 9 localities in that area and sequenced a 255-bp segment of the mtDNA D loop. Estimates of the minimum number of migration events from gene genealogies suggest that the linguistic barrier impaired gene flow more than the river did. However, an analysis of molecular variance (AMOVA) showed that most sequence diversity occurs within rather than between populations and that the differences between groups of populations, defined either by linguistic or geographic criteria, do not reach significance. Three areas of rapid genetic variation were identified; their locations suggest that populations of the western part of the study area evolved in relative isolation. Therefore mtDNA sequence variation does not seem to reflect the same processes--drift and presence of dispersal barriers--that led to the observed distributions of nuclear allele frequencies.