Hox genes encode a family of transcription factors that instruct positional identify along the anterior-posterior axis of animal bodies. The ‘posterior’ Hox group of vertebrates have undergone a deuterostome-specific expansion to pattern caudal and distal structures, like the motor neurons of the spinal cord. There, as elsewhere, Hox proteins with similar DNA-binding domains instruct a diversity of cell fates. Now, Milica Bulajić, Esteban Mazzoni and colleagues investigate how this occurs, using an in vitro motor neuron differentiation system. Expression of different Hox genes in progenitor motor neurons promotes distinct spinal cord fates, in a manner recapitulating embryonic development. ChIP-seq reveals distinct genome binding profiles for HOXC6, HOXC9 and HOXC10. In the case of HOXC9 and HOXC10, this is despite high homeodomain sequence similarity, and despite having similar sequence preference; rather, HOXC9 has a higher preference for relatively inaccessible chromatin. Furthermore, chromatin accessibility is increased to a large degree following HOXC9 binding. Even Hox proteins within the same HOX9 paralog group have distinct chromatin-binding capacities. HOXC13 genomic binding diverges from all other posterior groups because of a distinct sequence preference and chromatin binding profile. Finally, the genomic binding of posterior Hox proteins is associated with differential gene expression. Thus, divergence in posterior Hox patterning activity can be explained by sequence preference and how well they bind inaccessible chromatin.