The little skate, a cartilaginous fish evolutionarily distal from tetrapods, displays walking-like behavior and has conserved genetic programs and neuronal substrates for land-walking. Studies on little skate have been limited due to lack of high-quality genome assembly. Here, we generated an improved genome assembly of little skate reflecting precise gene annotation and structures and performed integrated analysis of gene expression and chromatin accessibility to investigate molecular mechanisms of fin motor neuron development. Through interspecies comparison of RNA expression, common and species-specific genes expressed in fin/limb/wing level motor neurons were identified. Moreover, by performing chromatin accessibility analysis with a pure fin motor neuron population the potential regulators controlling the gene expression in fin motor neurons were identified. Interspecies comparison of genomic data, gene expression, and chromatin accessibility assay suggest that the little skate has highly conserved gene regulatory mechanisms controlling tetrapod locomotion, which was not previously expected. Overall design: Examination of chromatin accessibility of little skate motor neuron compared with tail region spinal cord.