Purpose Prevalence estimates have been based on several case definitions of chronic fatigue syndrome (CFS). The purpose of this work is to provide a rigorous overview of their application in prevalence research. Methods A systematic review of primary studies reporting the prevalence of CFS since 1990 was conducted. Studies were summarized according to study design, prevalence estimates, and case definition used to ascertain cases. Results Thirty-one studies were retrieved, and eight different case definitions were found. Early estimates of CFS prevalence were based on the 1988 Centers for Disease Control and Prevention, Australian, and Oxford. The 1994 Centers for Disease Control and Prevention, however, has been adopted internationally, as a general standard. Only one study has reported prevalence according to the more recent, Canadian Consensus Criteria. Additional estimates were also found according to definitions by Ho-Yen, the 2005 Centers for Disease Control and Prevention empirical definition, and an epidemiological case definition. Conclusions Advances in clinical case definitions during the past 10 years such as the Canadian Consensus Criteria have received little attention in prevalence research. Future assessments of prevalence should consider adopting more recent developments, such as the newly available International Consensus Criteria. This move could improve the surveillance of more specific cases found within CFS.