The first retrospective molecular characterization of Mycobacterium tuberculosis from cerebrospinal fluid of 158 tuberculous meningitis (TBM) in Thailand, collected between 1995 and 2005, was performed by Southern-blot hybridization with an IS6110 probe and spoligotyping on 152 and 147 isolates, respectively. Antituberculous drug susceptibility testing was performed in 149 patients, 118 of whom (79.2%) contained pan-sensitive strains and eight (5.4%) harbored multidrug-resistant M. tuberculosis. IS6110 RFLP typing revealed 110 RFLP patterns with 57.9% of patients infected with the Beijing genotype. This percentage was significantly higher than that in a previous report from pulmonary tuberculosis patients. Fifteen of 18 TBM patients (83%) aged <15 years were infected with Beijing isolates (OR=4.47, p=0.018). There were 40 spoligotypes, with 118 patients (80.3%) being clustered. The biggest cluster, which consisted of 84 patients, was the Beijing spoligotype (57.1%). There were 16 novel spoligotypes from 16 patients compared to the Fourth International Spoligotyping Database, SpolDB4. Sixty-four percent of the patients were male, and the mean age of patients was 33.8 years. Beijing isolates from 2001 to 2005 were found in higher percentages than those from 1995 to 2000, but this difference was not significant (p=0.28).