The purpose of the present study was to determine the frequency of spontaneous axial skeletal variations detectable by the double staining technique in Crj: CD (SD) and Crj: Wistar rat fetuses. In addition, the frequencies of unossified skeletal variations were compared between SD and Wistar rats, and between same strain male and female rat fetuses. Fifteen types of the variations were detected. There were strain differences (4 types) and sex differences (3 types) in the frequencies of the variations. With the aid of double staining technique, skeletal variations such as supernumerary ribs or deformed ossification centers detected by the single staining technique could be classified in detail into many types. This study showed that the double staining technique improved detectability of skeletal variations and facilitated their evaluation in developmental toxicity studies.