Onuf's nucleus in the lumbosacral spinal cord, comprising somatic motoneurons that innervate the pelvic floor muscles via the pudendal nerve, shares some characteristics with the autonomic preganglionic neurons and functions in coordination with the autonomic nervous system. In mouse, neurons projecting to the urethral sphincter and ischiocavernosus muscles form the dorsolateral (DL) nucleus at the caudal lumbar levels, whereas neurons projecting to the limb and hip joint muscles comprise the retrodorsolateral and ventral nucleus, as well as the DL nucleus at the rostral lumbar levels. The results of the present study in mouse revealed that the expression pattern of a LIM homeodomein protein Islet-1, an embryonic marker for motoneurons in the spinal cord, was different among motoneuronal groups at the prenatal stage (embryonic days 13.5–15.5); the highest expression was observed in the DL at the caudal lumbar cord, whereas there was little expression in the lateral part of the rostral DL. Islet-1 expression was also observed in the parasympathetic preganglionic neurons at the sacral spinal cord. These findings provide evidence that the DL neurons at the caudal lumbar cord, corresponding to Onuf's nucleus, are chemically distinct among the motoneuronal groups at the prenatal stages. This differential Islet-1 expression among the motoneuronal groups suggests that Islet-1 not only leads to a motoneuronal lineage, but also to the differentiation of motoneuronal subsets in the lumbosacral spinal cord.