Motor neurons located at different positions in the embryonic spinal cord innervate distinct targets in the periphery, establishing a topographic neural map. The topographic organization of motor projections depends on the generation of subclasses of motor neurons that select specific paths to their targets. We have cloned a family of LIM homeobox genes in chick and show here that the combinatorial expression of four of these genes, Islet-1, Islet-2, Lim-1, and Lim-3, defines subclasses of motor neurons that segregate into columns in the spinal cord and select distinct axonal pathways. These genes are expressed prior to the formation of distinct motor axon pathways and before motor columns appear. Our results suggest that LIM homeobox genes contribute to the generation of motor neuron diversity and may confer subclasses of motor neurons with the ability to select specific axon pathways, thereby initiating the topographic organization of motor projections.