Tuberculosis is a major health problem throughout the world causing a largernnumber of deaths, more than that from any other single infectious disease. Extrarnpulmonary tuberculosis can occur in isolation or along with a pulmonary focusrnas in the case of patients with a disseminated form of tuberculosis. Tuberculousrnlymphadenitis (TBLN) is a common form of extra pulmonary tuberculosis.rnLymph nodes (cervical, axillary and inguinal) are the most common sites ofrninvolvement. Diagnosis of TBLN is a formidable challenge in developingrncountries where there is high rate of human immunodeficiency virus infection.rnThe aim of this study was to identify the etiological species of Mycobacteriarnresponsible for TBLN in Derra, rural Ethiopia, where the status of TBLN wasrnnot known. A total of 153 patients who fulfilled the inclusion criteria werernincluded in the present study during the study period between January 2004 andrnFebruary 2005. Out of 153 study participants, fine needle aspirate specimensrnfrom 145 study participants were processed and analyzed using Ziehl Neelsenrnstaining, culture, cytology and polymerase chain reaction. Out of 145 FNArnsamples, 66 (45.5%) of the isolates demonstrated growth on Lowenstein -rnJensen medium. Among 145 clinically suspected TBLN patients, 115 (79.3%)rnwere confirmed as TBLN cases by the combined results of AFB smearrnexamination, culture, cytology and PCR. Two primer sets were used to identifyrnthe etiological agent at the complex (targeting the IS6110 insertion sequence)rnand species level (targeting the pncA gene allelic variation). From a total of 145rnclinically suspected TBLN cases, 108 (75%) were positive by PCR at therncomplex levels. Based on PCR for detection of allelic variation at position 169,rn107/108 (99.1%) were positive for M. tuberculosis and only 1(0.9%) wasrnpositive for M. bovis. The results indicated that M. tuberculosis is the causativernspecies for tuberculous lymphadenitis in Dera.