Background: Using culture-based methods, bacterial translocation from the gut to draining mesenteric lymph nodes is seen in 5% of normal controls and up to 33% of patients with inflammatory bowel diseases (IBD). Many bacteria cannot be cultured, so these methods are unable to capture the full spectrum of bacteria present. Aims: To detect viable bacteria in lymph nodes of patients with IBD and non-IBD controls using bacterial RNA as a surrogate for viability and to compare them with the same patient's gut microbiome. Methods: Bacterial RNA was extracted from lymph nodes and mucosa of 20 patients who had undergone intestinal resection (10 IBD, 10 non-IBD). A previous study had detected bacterial DNA in these patients’ lymph nodes; 16S rRNA gene high-throughput sequencing was performed. Results: Bacterial RNA was detected in the lymph nodes of five patients with IBD and three controls (volvulus, diverticulitis and bowel obstruction). Lymph nodes had higher alpha (within sample) diversity compared to mucosal samples (Shannon diversity index 2.41 vs 1.81, one-way ANOVA P = 0.035). Beta diversity (inter-sample variation: lymph node vs intestine) was similar within individuals and did not differ between groups. Common gene polymorphisms linked with IBD (NOD2, ATG16L1, IRGM and IL23R) were not associated with bacterial translocation. Conclusions: Metabolically active bacteria mirroring the individual's gut microbiome were commonly found in the lymph nodes of patients with IBD undergoing resection. An increase in lymph node alpha diversity is likely due to the larger drainage area. The presence of viable bacteria in non-IBD controls is also not unexpected given the underlying pathology in these patients.