Multidrug-resistant tuberculosis (MDR-TB) is a cause of increasing concern. This study investigated first-line anti-TB drug resistance in Mycobacterium tuberculosis strains submitted to the Tuberculosis Reference Center in Córdoba (Spain) between 2001 and 2015. A total of 1,207 cultures were tested against first-line drugs using the BACTEC MGIT 960 system. Resistance to first-line drugs was detected in 207 strains (17.2%), the greatest resistance being found in INH (5.3%) followed by streptomycin (3%), pyrazinamide (2.2%), rifampicin (1%), and ethambutol (0.2%). A total of 1.9% of strains were MDR-TB. Six strains displayed resistance to four drugs, and three strains to five drugs. In view of resistance observed, careful surveillance of drug resistance is recommended.