We investigated whether the installation of sluice gates hindered gene flow among subpopulations of a fluvial lamprey, Lethenteron sp. N, inhabiting a paddy water system. Individuals were collected from three study sites and were genotyped at six polymorphic microsatellite loci. Our calculations indicated that, historically, gene flow among the subpopulations was frequent and bidirectional; however, contemporary gene flow was unidirectional (upstream to downstream). This indicates that the sluice gates have obstructed the movement of individuals between different subpopulations. Despite this, genetic diversity was similar across all three study sites. Thus, while the fluvial lamprey population does not appear to be in danger of losing genetic diversity, continued isolation is likely to affect gene flow by reducing migration between subpopulations. We recommend a management scheme wherein the sluice gates are periodically opened in order to facilitate migration. Since lampreys are an important part of the ecosystem, this should also help maintain the health of the paddy environment in general.