Objectives: Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a disabling illness characterised by persistent fatigue and a multitude of symptoms. CFS/ME symptom presentation varies greatly among patients, suggesting potential severity subgroups within the illness. Studies have also only consistently focused only on CFS/ME patients with moderate severity. Therefore this study investigated CFS/ME severity subgroups in CFS/ME while assessing a wide range of innate (NK, neutrophil, monocyte and DC’s) and adaptive (iNKT and B cells) immune cells based on phenotypes and functioning to determine the extent of immunological dysfunction. Methods: Participants were grouped into 22 non-fatigued controls (age = 40.14 + 2.38), 23 moderate (age = 42.52 + 2.63) and 18 severely (age = 39.56 + 1.51) affected CFS/ME patients using a number of severity scales. Results: Results confirmed significant decreases in NK cytotoxic activity and alterations in NK, DCs, iNKT and B cell phenotypes. Conclusion: This is the first to determine alterations in DC, iNKT and B cell phenotypes in severe CFS/ME patients. This supports the notion that immunological biomarkers are necessary in CFS/ME and that severity subgroups should be assessed in both clinical and research settings.