We conducted a meta-analysis of studies comparing the presence of Chlamydia pneumoniae (Cpn) between multiple sclerosis (MS) patients and other neurological diseases patients or healthy controls. We identified 26 studies with 1332 MS patients and 1464 controls. Using random-effects methods, MS patients were found more likely to have detectable levels of Cpn DNA (OR = 3.216; 95% CI: 1.204, 8.585) in their cerebrospinal fluid, and intrathecally synthesized immunoglobulins (OR = 3.842; 95% CI: 1.317, 11.212), compared to other patients with neurological diseases. There is no evidence for increased levels of serum immunoglobulins (OR = 1.068; 95% CI: 0.745, 1.530), even though this result is confounded by the presence of studies using normal subjects as controls. Similarly, there is no evidence for association of immunoglobulins against Cpn in the cerebrospinal fluid (OR = 3.815; 95% CI: 0.715, 20.369). Up to 59.7% of the between-studies variability could be explained by the inappropriate matching of cases and controls for gender. In random-effects meta-regressions, adjusting for the confounding effect of gender differences results in stronger and statistically significant associations of MS with detectable levels of Cpn DNA, intrathecally synthesized immunoglobulins and immunoglobulins in the cerebrospinal fluid. Even though the presence of Cpn is clearly more likely in MS patients, these findings are insufficient to establish an etiologic relation.