Multidrug resistant Mycobacterium tuberculosis (MDR-TB) is of great public health importance worldwide. This three month laboratory- based study (1st September-30th November, 2011) was carried out at the TB laboratories of the University College Hospital, Ibadan, Nigeria to determine the magnitude of MDR-TB using molecular based GenoType MTBDRplus 96 assay. Two sputum samples were collected from each subject. These were processed using Ziehl -Neelsen (ZN) reagents. The sputa were cultured on Loewenstein-Jensen egg -based medium and incubated at 370C for eight weeks. Mycobacterium tuberculosis complex (MTBC) was confirmed by colonial morphology and repeat ZN staining. All the Acid-fast bacill (AFB) positive smears and culture positive isolates were tested for genetic identification and drug susceptibility testing (DST) using PCR- based GenoType MTBDRplus 96 assay (HAINs Lifesciences, GmbH, Nehren, Germany) according to manufacturers' instructions. Of the 68 samples processed, 11 (16.2%) were AFB positive while six (8.8%) were positive for culture. Eleven (64.7%) out of the 17 samples tested for genetic identification were MTBC while six were Non Tuberculosis Mycobacteria (NTM). All NTM were from AFB positive sputum while none was from culture positive isolates. Of the six culture isolates tested for DST, three (33.3%) were susceptible to isoniazid and rifampicin; one (16.7%) showed mono-resistance to isoniazid while two (30.0%) were resistant to the two drugs. This study shows that MDR-TB is present in Ibadan. There is a need to make DST diagnostic facilities more available and accessible in Nigeria.