BACKGROUND: Drug resistance surveys have not yet conducted in these Islands and as such no data exists on drug resistance currently. AIMS: the present study was initiated with the objective of isolation and assessment of Drug resistance patterns of Mycobacterium tuberculosis isolates from sputum specimens collected from different categories of Tribal, Non-Tribal pulmonary tuberculosis patients treated under DOTS and Non-DOTS program by conventional culture and Proportion sensitivity (PST) method to detect patients with Multidrug resistant strains. METHODS: The investigation was hospital based laboratory surveillance study carried out for a period of 3 years at the selected hospitals of Andaman district (TB ward GB Pant Hospital at Port Blair, CHC Bamboflat at Port Blair and CHC Rangat at Rangat) and Nicobar district (CHC Nancowry at Nancowry groups of Islands), among the new cases and re-treatment cases of tuberculosis patient under DOTS program and Non-DOTS patients attended selected hospitals of Andaman & Nicobar districts chosen for the study. RESULTS: 83 culture positive isolates obtained (74 identified as M. tuberculosis) from the sputum specimen of 162 cases of tuberculosis patient by conventional culture method. 60 M. tuberculosis isolates subjected to drug susceptibility test using PST method, 7 patients (11.67%) found to be Multidrug resistant tuberculosis (MDR-TB), resistant patterns were S + H + R + E = 1(Cat II-DOTS),H + r = 3(Cat-I DOTS = 1, Cat II-DOTS-1,Non-DOTS = 1), Rifampicin resistant alone = 2 (Non-DOTS = 1, Cat II-DOTS = 1) and R + E = 1(Cat I-DOTS). CONCLUSIONS: Laboratory finding suggested that nine MDR-TB strains detected in DOTS and Non-DOTS among 60 M. tuberculosis isolates were selected for drug susceptibility testing but two isolates detected as MDR-TB from patients was already on Second line drugs treatment were not included in the MDR-TB detection criteria. Hence 7 patients (11.67%) declared to be Multidrug resistant tuberculosis (MDR-TB). 2 MDR-TB strains with resistant patterns H + r = 1(Cat II-DOTS), Rifampicin resistant alone = 1(Non-DOTS) detected from 12 isolates of Tribal patients from Nicobar district and 5 MDR-TB strains with resistant patterns S + H + R + E = 1 (Cat II-DOTS), H + r = 2(Cat I-DOTS = 1,Non-DOTS = 1), Rifampicin resistant alone = 1 (Cat II-DOTS) and R + E = 1(Cat I-DOTS) detected from 48 isolates of Non-Tribal patients from Andaman district. To assess the MDR-TB burden in the islands, systematic drug resistant surveillance study needs to be conducted.