Limited immunological changes have been previously reported in B cell phenotype in Chronic Fatigue Syndrome (CFS) patients, so there is no clear established role of B cells in the pathophysiology of CFS patients. The aim of this study was to evaluate B cells subsets including naive, memory naive, memory switched, memory non-switched, double negative, transitional, plasmablasts, HLA-DR+, plasma and regulatory B cells (Breg) in CFS patients compared with non-fatigued controls. B cell activation markers (CD81, CD21) and surface receptors (CD79a/b, IgM, IgD, IgA, IgE) were also examined in CFS patients compared with non-fatigued controls. 46 CFS patients (age=50.00 ± 2.00 years) and 34 non-fatigued controls (age=49.00 ± 2.16 years) participated in the study. The percentage of BCR IgM+ B cells was significantly increased in the CFS group compared with non-fatigued controls (p=0.037). Similarly, there was a significant decrease in the CD1d+ B cells in the CFS group compared with nonfatigued controls (p=0.046). No additional differences in B cell phenotypes, activation markers and surface receptors were found in the CFS patients compared with the non-fatigued control group. The differences observed in the B cell phenotype of CFS patients compared with non-fatigued controls may explain some of the disturbances in the immune homeostasis, however whether this is causal or the consequence of immunological imbalances previously reported in CFS patients requires further investigation.