In Spain, tuberculosis (TB) patterns are changing because of the recent increase in the number of cases among immigrants. To establish the composition of circulating Mycobacterium tuberculosis strains before the effects of foreign strains appear, this study focused on molecular characterization of 233 patient isolates using spoligotyping. The spoligotyping data were further analyzed using an international database, SpolDB4. The results obtained showed that the general features of the M. tuberculosis population in Spain are coherent with those of other European countries, with the Latin American and Mediterranean group, and with the Haarlem 3 and T1 families as the most prevalent genotypes. The Spanish isolates clustered mostly with genotypes which had previously been isolated in countries linked with Spain. We also describe and fully characterize two novel M. tuberculosis families, Madrid1 and Madrid2, which are specific to Spain-related settings. The data reported here provide a solid reference when monitoring changes in the composition of the M. tuberculosis population in Spain as a consequence of the increasing rate of TB in the foreign population.