To elucidate the petromyzontid speciation process, the genetic independence of the fluvial non-parasitic populations within the anadromous parasitic Lethenteron camtschaticum was estimated by using polymorphic microsatellite loci. Abundant gene flow was revealed in multitemporal scales between potentially sympatric populations, suggesting ongoing gene flow resulting from imperfect size-assortative mating between them and plastic determination of life histories. On the contrary, landlocked fluvial non-parasitic populations in the upper region of dams were genetically divergent from anadromous parasitic populations. The temporal heterogeneity of gene flow, i.e. contemporary little gene flow but significant gene flow over the long-term between the landlocked fluvial non-parasitic and anadromous parasitic populations was elucidated. In addition, the divergence time of isolation of the landlocked populations from the ancestral anadromous parasitic population was estimated to have occurred 17·9–428·2 years ago, which includes the construction times of an initial dam c. 90 years ago. These instances indicate that the landlocked populations should have very recently been established, and subsequent accumulation of divergence and development of reproductive isolation are predicted. The present landlocked fluvial non-parasitic populations should be analogous to the founder populations in terms of petromyzontid speciation. The data also strongly support the hypothesis of multitemporal and multispatial speciation in the petromyzontid stem–satellite species complex.