In recent years, the percentage of tuberculosis (TB) cases in immigrants in Spain has increased markedly. In this context, discrimination between cases with potentially imported TB and cases likely to have acquired it by recent transmission after arrival is a basic issue. In this study, we evaluated molecular markers to obtain information on the geographic origin of Mycobacterium tuberculosis (MTB) strains and to determine whether they could help to identify imported TB cases. We analyzed the pks15/1 gene, spoligotype, and MIRU locus 24 in 168 MTB isolates from immigrants of 32 nationalities and from a selection of autochthonous cases. In non-Asian immigrants we could not detect major differences with the autochthonous cases. For the Asian cases, we found some specific features for these markers. pks15/1 was intact in 69% of the Asian patients, but this gene had a 7-bp deletion in all non-Asian and Spanish (non-Beijing) cases. The spoligotype-defined lineages EAI and CAS, and the allele with two repetitions in MIRU locus 24 was found exclusively among Asian immigrants. The analysis of certain bacterial molecular markers could help to discriminate between potentially imported TB cases and those more probably acquired in the host country.