The aim was to compare clinical characteristics, antimicrobial resistance and 24-loci variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) patterns among M. tuberculosis complex isolates (MTBC) recovered from patients living in Greece during a two-year period (2009-10). Isolates from 117 tuberculosis patients identified as MTBC by a molecular method were phenotypically tested for their antimicrobial susceptibility. Clonality was accessed by MIRU-VNTR. Among 117 patients with confirmed microbiologic tuberculosis, 90 (76.9%) were of Greek origin, and 27 (23.1%) foreigners. Only 67 out of the 117 (57.3%) patients were officially reported to the Hellenic Center for Disease Control and Prevention. Phenotypic susceptibility testing revealed 10 isolates resistant to isoniazid (8.5%), two to rifampicin (1.7%), five to ethambutol (4.3%) and 17 to streptomycin (14.5%). In total two multidrug-resistant strains (1.7%) were detected. MIRU-VNTR classified 35 strains (29.9%) into 10 Families, whereas, 82 (70.1%) were not classified into any known Family. MTBC strains belonging to known Families were more commonly isolated from people living in cities and foreigners and showed a trend to higher resistance percentages to isoniazid and rifampicin. Most isolates from Greek rural areas do not belong to any known Family and are characterized by lower resistance rates.