Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. Thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia were collected from 10 mice 3-10 days after the neuronal tracer Fast Blue was injected into the wall of the colon via laparotomy under inhalation anaesthesia (1.25 % isoflurane) following previously described protocols (Hockley et al, 2014). Fast Blue labelled mouse colonic sensory neurons were subsequently picked using an adapted patch-clamp rig and pulled glass pipettes, and single-cell RNA-sequencing (Picelli et al, 2013) was performed on cells isolated from both TL (n=159 neurons) and LS (n=155 neurons) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes using Single-cell Consensus Clustering (SC3, Kiselev et al, 2017). The subtypes were validated by performing single-cell qRT-PCR on a second cohort of 168 colonic sensory neurons against a panel of 7 subtype-specific marker genes (Mrgprd, Cbln2, Fam19a1, Smr2, Trpa1, Hpse and Ntm). We also used immunohistochemistry (IHC) against a panel of markers including TrkC, Gfra3 and Spp1 to validate the spinal segmental patterning observed between TL and LS neurons. Of the seven subtypes identified, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We use Ca2+-imaging of mouse colonic sensory neurons combined with post-functional single-cell qRT-PCR to demonstrate subtype-selective differential agonist activation (e.g. 10 of 125 TL neurons responded to 5-HT4 receptor agonist BIMU8 but no LS neurons (0 of 42) matching the expression profile of 5-HT4 within TL only subtypes). These results provide a framework to the molecular interrogation of colonic sensory innervation in health and disease and also an opportunity to identify novel targets for future drug development.