Sensorimotor circuits of the lumbosacral spinal cord are required for lower urinary tract (LUT) regulation as well as being engaged in pelvic pain states. To date, no molecular markers have been identified to enable specific visualization of LUT afferents, which are embedded within spinal cord segments that also subserve somatic functions. Moreover, previous studies have not fully investigated the patterning within or across spinal segments, compared afferent innervation of the bladder and urethra, or explored possible structural sex differences in these pathways. We have addressed these questions in adult Sprague-Dawley rats, using intramural microinjection of the tract tracer, cholera toxin B subunit. Afferent distribution was analysed within individual sections and 3D reconstructions from sections across four spinal cord segments (L5-S2), and in cleared intact spinal cord viewed with light sheet microscopy. Simultaneous mapping of preganglionic neurons showed their location throughout S1 but restricted to the caudal half of L6. Afferents from both LUT regions extended from L5 to S2, even where preganglionic motor pathways were absent. In L6 and S1, most afferents were associated with the sacral preganglionic nucleus and sacral dorsal commissural nucleus, with very few in the superficial laminae of the dorsal horn. Spinal innervation patterns by bladder and urethra afferents were remarkably similar, likewise the patterning in male and female rats. In conclusion, micro- to macro-scale mapping has identified distinct features of LUT afferent projections to the lumbosacral cord and provided a new anatomical approach for future studies on plasticity, injury responses and modelling of these pathways.Significance statementIn this multi-scale neuroanatomical study, we have developed novel maps of bladder and urethra afferent pathways in the spinal cord of male and female rats. Afferents visualised by transganglionic tracing were registered to anatomical landmarks including motoneuron pools and preganglionic autonomic neurons, enabling comparisons across animals and specific regions of the cord. We integrated data from 3D reconstructions of sections, light sheet microscopy of cleared, contiguous spinal segments and confocal microscopy to assess connectivity at microscale. In addition to providing new understanding of lumbosacral visceral circuitry, this dataset provides the foundation for future studies on plasticity and regenerative capacity of these circuits, changes during the life cycle and parameters for modelling spinal circuits relevant to lower urinary tract function.