Emotional reactivity to negative stimuli has been investigated in insomnia, but little is known about emotional reactivity to positive stimuli and its neural representation. We used 3 Tesla functional magnetic resonance imaging (fMRI) to determine neural reactivity during the presentation of standardized short, 10- to 40-seconds, humorous films in patients with insomnia (n = 20, 18 females, aged 27.7 +/- 8.6 years) and age-matched individuals without insomnia (n = 20, 19 females, aged 26.7 +/- 7.0 years) and assessed humor ratings through a visual analog scale. Seed-based functional connectivity was analyzed for the left and right amygdalas (lAMYG and rAMYG, respectively) networks: group-level mixed-effects analysis (FLAME; FMRIB Software Library [FSL]) was used to compare amygdala connectivity maps between groups. fMRI seed-based analysis of the amygdala revealed stronger neural reactivity in patients with insomnia than in controls in several brain network clusters within the reward brain network, without humor rating differences between groups (p = 0.6). For lAMYG connectivity, cluster maxima were in the left caudate (Z = 3.88), left putamen (Z = 3.79), and left anterior cingulate gyrus (Z = 4.11), whereas for rAMYG connectivity, cluster maxima were in the left caudate (Z = 4.05), right insula (Z = 3.83), and left anterior cingulate gyrus (Z = 4.29). Cluster maxima of the rAMYG network were correlated with hyperarousal scores in patients with insomnia only. The presentation of humorous films leads to increased brain activity in the neural reward network for patients with insomnia compared with controls, related to hyperarousal features in patients with insomnia, in the absence of humor rating group differences. These novel findings may benefit insomnia treatment interventions. The Sleepless Brain: Neuroimaging Support for a Differential Diagnosis of Insomnia (SOMNET). ClinicalTrials.gov identifier: NCT02821234; https://clinicaltrials.gov/ct2/show/NCT02821234.Read less <