Vagal afferent neurons (VAN) provide information about meals to inhibit food intake (FI). The role of VAN in mediating the effects of different macronutrients remains unclear. To address whether macronutrients activate separate vagal populations, we first adapted a technique for in vivo imaging of nodose ganglia neurons using mice expressing the Ca2+ indicator GCaMP6s driven by the pan-neuronal promoter Snap25. We found that separate neuronal populations were activated in response to intragastric infusions of fat or sugar. Next, in order to genetically access NG neurons that were active in response to post-ingestive fat or sugar sensing, we used a transgenic mouse line that allows targeted recombination in active populations (TRAP). These mice express an inducible cre recombinase, iCreERT2, under the control of an activity-dependent c-Fos promoter (FosTRAP mice), enabling permanent genetic access to neuronal populations based on their activation to a defined, time-constrained stimulus when paired with injection of 4-hydroxytamoxifen. To assess whether stimulation of fat or sugar responsive vagal neurons are each independently sufficient for reward, mice underwent a previously-validated self-stimulation behavioral task in which optogenetic stimulation of vagal sensory terminals is paired to nose poke. We found that mice learned to self-stimulate for optogenetic stimulation of vagal sensory neurons that are activated in response to fat or sugar. To assess the necessity of separate macronutrient-sensitive vagal sensory populations for food reinforcement, we performed a flavor-nutrient conditioning task, in which animals are trained to prefer a novel flavor that has been experimentally-paired to intragastric infusion of nutrient. Selective deletion of fat or sugar sensing vagal neurons prevents nutrient-specific reinforcement. Thus, post-ingestive sensing of fat and sugar are both necessary and sufficient for the development of macronutrient-specific reward. Next, we inquired whether post-ingestive signals diverge at the cellular level along the well-defined gut reward circuit. We used FosTRAP mice to address the challenge of comparing neuronal activity in response to multiple stimuli at different timepoints over multiple brain regions in the same mouse. When analyzing the overlap between sucrose TRAP and sucrose Fos labeling, we found >60% overlap in the nucleus tractus solitarius, parabrachial nucleus, Substantia nigra pars compacta and dorsal striatum. However, when comparing sucrose TRAP and fat Fos labeling, there was only limited overlap between the neurons labeled during the two separate nutritive stimuli.