The default mode network (DMN) has been proposed as a biomarker for several chronic pain conditions. DMN functional connectivity (fcMRI) is typically examined during resting-state fMRI, in which participants are instructed to let thoughts wander. However, factors at the time of data collection (e.g., negative mood) that might systematically impact pain perception and its brain activity, influencing the application of the DMN as a pain biomarker, are rarely reported. The present study measured whether positive and negative moods altered DMN fcMRI patterns in chronic low back pain (CLBP) patients, specifically focusing on negative mood due to its clinical-relevance. Thirty-three participants (CLBP = 17) underwent resting-state fMRI scanning before and after sad and happy mood inductions, and rated levels of mood and pain intensity at the time of scanning. Two-way repeated measures ANOVAs were conducted on resting-state functional connectivity data. Significant group (CLBP > HC) X condition (sadness > baseline) interaction effects were identified in clusters spanning parietal operculum/postcentral gyrus, insular cortices, anterior cingulate cortex, frontal pole, and a portion of the cerebellum (pFDR < .05). However, only one significant cluster covering a portion of the cerebellum was identified examining a two-way repeated measures ANOVA for happiness > baseline (pFDR < .05). Overall, these findings suggest that DMN fcMRI is affected by negative mood in individuals with and without CLBP. It is possible that DMN fcMRI seen in chronic pain patients is related to an affective dimension of pain, which is important to consider in future neuroimaging biomarker development and implementation.