Background:- Tuberculosis is the leading cause of death in hospitalized patients. It has notrnyet been investigated whether MDR-TB contributes to the reported high mortality. Therernhas also been no study on the molecular epidemiology of TB and M. tuberculosis strainrntypes have not yet been characterized in the area. Nationwide, there have been no previousrnreports on drug resistance in individuals treated with 4 fixed dose combination (FDC) anti-rnTB drugs. Shashemene is one of the known locations for high prevalence of TB and HIV inrnEthiopia. The TB-HIV co-infection rate in the age group of 15-49 years was estimated tornbe 47% in the area (MoH, 2004).rnObjectives:-To determine the prevalence of resistance to the four first line anti-TB drugsrnwith and without HIV co-infected TB patients and to investigate association of TB bacteriarnstrain types with drug resistance.rnMethods: - A cross-sectional survey on anti-TB drug resistance was conducted in fourrnhealth centres and one Hospital in and around Shashemene in Ethiopia. Sputum and bloodrnsamples were collected from all consenting smear positive pulmonary TB patients visitingrnthe sites between May 2006 and December 2006. Sputum was digested and decontaminatedrnusing Petroff s method with 4% NaOH and cultured on Lowenstein Jensen media. Drugrnsensitivity tests were performed on isolates using the proportion method on Middlebrookrn7H10 media and 10% OADC enrichment. Pateints were screened for HIV with rapid assaysrn(DetermineÂ®, CapillusÂ® and UnigoldÂ®) according to national guidelines. Species werernidentified with biochemical (Thiophene-2-Carboxylic acid Hydrazide TCH test) andrnDNA based methods (species specific PCR amplification with RD4 primer). Drug resitancernwas further characterized using PCR based mutation analysis as a rapid and simplerntechnique for diagnosis of drug resistance in tuberculosis. We described a simple multiplexrnallele-specific (MAS)-PCR assay to detect mutations in the second base of the katG generncodon 315, including AGC ACC and ACA (Ser Thr) substitutions that confer resistancernto isoniazid (INH) in M. tuberculosis clinical isolates. We used PCR-RFLP assay forrnrpsL43 mutation analysis, in the absence of mutation in AAG AGG position in MboIIrndigest rpsL43 and the fragment was a 210-bp and 60-bp. If a mutation in codon 43 resultedrnxiiirnin a 270-bp fragment.rnResults and Discussion: - A total of 292 (264 new and 28 previously treated) patients werernincluded in this study. Out of these, 82.5% (241/264) were culture positive. Sensitivity testrnresults were available for 217 isolates from new cases and 24 isolates from previouslyrntreated patients. Among the isolates, 80% (174/217) of those from new patients and 54.2%rn(13/24) of those from previously treated patients were sensitive to all drugs tested.rnPrevalence of MDR-TB among new cases was 0.9% (2 isolates). Resistance to INH, RMP,rnSTM and EMB was 12.9% (28/217), 1.8% (4/217), 16.6% (36/217) and 4.1% (9/217)rnrespectively. In previously treated patients INH, RMP, STM and EMB resistance wasrn37.5% (9/24), 8.3% (2/24), 41.7% (10/24) and 12.5% (3/24) respectively. There was alsornno association observed between drug resistance among new cases and HIV. The highrnprevalence of the katG315 ACC mutant allele among INH resistant and rpsL43 AGGrnmutant allele among STM resistant M. tuberculosis clinical isolates in the Shashemenernarea, Southeast Ethiopia, 83.8% (31/37) and 55.56% (20/36) for all isolates was observed.rnConclusion: Prevalence of Multi-drug resistant TB in the Shashemene area is comparablernwith the national report. M. bovis was not isolated from pulmonary TB in the area.rnKey words: Proportion methods; Multi-drug resistant tuberculosis; Tuberculosis; HumanrnImmunodeficiency Virus; Analysis of TB drug resistant mutants;