Neural circuits in the spinal cord are composed of diverse sets of interneurons that play a crucial role in shaping motor output. Despite progress in revealing the cellular architecture of the spinal cord, the extent of cell type heterogeneity within cardinal interneuron classes remains unclear, hindering our understanding of the organization of the spinal motor infrastructure. Through a focus on a single cardinal class of spinal interneurons, here we present a comprehensive atlas of V1 interneuron diversity across postnatal development using single-nucleus transcriptomics. We find that cell-type diversity is largely preserved across postnatal development, providing insight into the molecular taxonomy that distinguishes both known and novel V1 clades from birth to adulthood. Interestingly, the transcription factor Engrailed-1 (En1), which delineates the parental V1 population, plays a critical role in the development of a highly restricted V1 subset while sparing the vast majority of V1 interneurons. En1 global and conditional knock-out mice exhibit slowed locomotor rhythm but do not exhibit hyperflexion, suggesting a dissociation between these two previously defined phenotypes that are observed upon ablation or inhibition of the larger V1 population. Beyond serving as a molecular resource for this neuronal population, our study highlights how deep neuronal profiling provides an entry point for the study of rare, specialized cell types in the spinal motor system.