Field-collected specimens of glossiphoniid leeches, Torix tagoi, Torix tukubana, Hemiclepsis marginata, and Hemiclepsis japonica, were surveyed for Rickettsia infection by using a diagnostic PCR assay. Rickettsia was detected in 96% (69/72) of T. tagoi, 83% (24/29) of T. tukubana, 29% (33/113) of H. marginata, and 0% (0/30) of H. japonica. The frequencies of Rickettsia infection were stably maintained in different seasons. In H. marginata and T. tukubana, distant local populations exhibited remarkably different frequencies of Rickettsia infection. Eggs carried by infected females of T. tagoi and H. marginata were all Rickettsia-positive, indicating nearly 100% vertical transmission. Analysis of 16S rDNA sequences revealed that phylogenetic relationship of the leech-associated Rickettsia reflected the specific and populational divisions of the host leeches. However, circumstantial lines of evidence strongly suggested that horizontal transmission of Rickettsia must have occurred in the ancestors of these leeches. In T. tagoi and T. tukubana, infected individuals were remarkably larger in size than uninfected individuals, wheras in H. marginata, infected and uninfected individuals were almost comparable in size. This study first provides information on ecological aspects of leech-bone endocellular bacteria of the genus Rickettsia. On the basis of these data, we discuss possible mechanisms whereby Rickettsia infection is maintained in natural populations of these leeches in the freshwater ecosystem.