Preferred habitat condition of the spined loach, Cobitis matusbarae (Cobitidae) was surveyed at the Saigo River, Fukuoka Prefecture, Kyushu Island, Japan, between immature and adult fish. The studied small stream is 140m long flowing through Fukutsu City, and located 4 km stream from the river mouth. The number of C. matusbarae individuals was checked, and 10 physical environmental parameters were measured to assess the microhabitat in 45 quadrates. The number of individuals was counted each month, and environmental measurements were carried out 4 times from January to June 2010. Akaike’s Information Criterion (AICc) and Generalized Linear Model (GLM) were utilized for analysis to verify the effect of the important environmental variables on the habitat of the C. matusbarae. A total of 184 adults and 15 juveniles were captured. The adult populations correlated positively with the water depth during the non-spawning season and with short emergent hydrophytes during the spawning season. Juveniles preferred shallow temporary water around short emergent hydrophytes which was spawning site of the adults.