From April 2008 to July 2009, an acoustic telemetry study on crucian carp was conducted in Lake Biwa, Japan. Twenty Nigorobuna, ten gengoroubuna and one ginbuna captured during spawning seasons were released with ultrasonic transmitters in the South Basin. Signals from the tagged fish were detected by 28 ultrasonic-receiver stations installed around Lake Biwa. Seasonal preference of habitats was examined by distance-based analysis. Seasonal distributions of nigorobuna and gengoroubuna in Lake Biwa were observed including consecutive two spawning seasons. It was revealed that seasonal migration patterns of nigorobuna and gengoroubuna were different. Nigorobuna appeared mainly around the release site all the year round, while gengoroubuna migrated to the North Basin after their spawning season, where they stayed during the non-spawning season, and moved to the South Basin in the next midwinter, where they stayed until the end of the next spawning season. It was also revealed that gengoroubuna preferred lower water temperature than nigorobuna. Gengoroubuna moved faster than nigorobuna and ginbuna. There were high-degree correlations in distance ratios of each station during spawning seasons in the release year and the next year in both nigorobuna and gengoroubuna, suggesting a multiyear use for the same spawning site. Through these results, deeper understanding of these endangered fish species in Lake Biwa and information to help the development of effective conservation programs would be provided.