Dissimilatory Iron-Reducing Bacteria (DIRB) are known to produce magnetite in medium with amorphous iron oxide such as ferrihydrite that have been isolated from various sites including freshwater and marine sediments in the world. However, the isolation of DIRB from freshwater in Japan has not been reported. We attempted to detect DIRB from freshwater sediments collected at four sites in Shizuoka Prefecture using ferrihydrite-enriched cultures and PCR-denaturing gradient gel electrophoresis (DGGE) analysis. After 14 days of incubation, the ferrihydrite medium turned black and showed a magnetic response in vials containing the all samples. In the DGGE profiles, characteristic bands could be determined in all samples before and after incubation, and significant changes occurred in the microbial community after incubation in the ferrihydrite-enriched cultures. The main DGGE bands in the four samples after incubation showed 98% similarity with Bacterium ROME215Asa, Geobacter thiogenes K1 (98%), Geobacter sp. T32 (100%), and Geobacter sulfurreducens PCA (100%), respectively. These results indicate that ferrihydrite-enriched cultures coupled with PCR-DGGE analysis are an effective means of detecting DIRB in the environment. Furthermore, this study revealed that several species of DIRB exist with various kinds of bacteria in freshwater sediments in Shizuoka Prefecture, Japan.