More rapid, and specific molecular diagnostic methods are required to understand the epidemiology of tuberculosis in the population and to establish effective control measures. Molecular epidemiologic studies about Mycobacterium tuberculosis are yet limited in Turkey. Cukurova (Eastern Mediterranean part of Turkey) region is of special importance in terms of tuberculosis epidemiology due to its neighbourhood countries and relatively high number of immigrants to that area. This study was aimed to determine the genotypic characteristics of M.tuberculosis strains isolated from pulmonary tuberculosis patients in Cukurova region, by spoligotyping and 12 loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats) methods. A total of 467 M.tuberculosis strains isolated from patients with pulmonary tuberculosis admitted to 20 hospital/dispansery in seven different provinces (Adana, Mersin, Osmaniye, Antakya, Gaziantep, Sanliurfa, Kahramanmaras) at Cukurova region between January 2007-June 2010, were included to the study. Genotypic identification was done by spoligotyping and 12 loci MIRU-VNTR methods. M.tuberculosis complex strains were isolated from different samples (sputum, bronchoalveolar lavage and biopsy material) by MGIT 960-TB (BD Diagnostics, USA) liquid culture method in Regional Tuberculosis Laboratory. Spoligotyping analysis revealed that 443 (94.9%) of the strains were clustered in 21 groups while 24(5.1%) of the isolates were described as orphan strains according to SpolDB4 database. The most common families were T1 genotype seen in 239 (51.9%) strains and LAM7 TUR genotype seen in 54 (11.5%) strains. Six (1.3%) strains isolated from one province were multidrug resistant strains and belonged to Beijing family. Combination of spoligotyping with 12-locus MIRU-VNTR divided the T1 family into 7 clusters of which members ranged from 2-158. Twenty one isolates showed unique pattern. According to this study, T1 family is the most common genotype among M. tuberculosis strains in Cukurova, Turkey and the prevalence of M. tuberculosis Beijing strains was 1.3%, detected only in one province (Sanliurfa). Active surveillance studies are necessary to follow the regional dissemination of M.tuberculosis genotypes and establish effective precautions to prevent the spread of especially drug resistant strains.