Mapping the expression of transcription factors in the mouse spinal cord has identified ten progenitor domains, four of which are cardinal classes of molecularly-defined, ventrally located interneurons that are integrated in the locomotor circuitry. This review focuses on the properties of these interneuronal populations and their contribution to hindlimb locomotor central pattern generation. Interneuronal populations are categorized based on their excitatory or inhibitory functions and their axonal projections as predictors of their role in locomotor rhythm generation and coordination. The synaptic connectivity and functions of these interneurons in the locomotor central pattern generators (CPGs) have been assessed by correlating their activity patterns with motor output responses to rhythmogenic neurochemicals, sensory and descending fibers stimulations as well as analyzing kinematic gait patterns in adult mice. The observed complex organization of interneurons in the locomotor CPG circuitry, some with seemingly similar physiological functions, reflects the intricate repertoire associated with mammalian motor control, and is consistent with high transcriptional heterogeneity arising from cardinal interneuronal classes. The review discusses insights derived from recent studies to describe innovative approaches, limitations in experimental model systems, and identify missing links in current investigational enterprise.