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Tsehaye Asmelash Dejene,1,2 Genet Gebrehiwet Hailu,1 Atsebaha Gebrekidan Kahsay,1 Araya Gebreyesus Wasihun1 1Department of Medical Microbiology and Immunology, School of Medicine, College of Health Sciences, Mekelle University, Mekelle, Ethiopia; 2Department of Medical Microbiology, School of Medicine, Aksum University, Axum, EthiopiaCorrespondence: Atsebaha Gebrekidan Kahsay, Department of Medical Microbiology and Immunology, School of Medicine, College of Health Sciences, Mekelle University, P. O. Box: 1871, Mekelle, Tigrai, Ethiopia, Email atsebaha.gebrekidan@mu.edu.etBackground: Tuberculosis (TB) continues to be a global health problem. While childhood TB contributes 10% to the global TB burden, the paucibacillary nature of TB disease in children and the absence of reliable diagnostic methods have made MTB diagnosis in children to be a great challenge. This study aimed to determine the prevalence of MTB and rifampicin-resistant MTB (RR-MTB) among children using Gene Xpert MTB/RIF Assay in Tigray, Ethiopia.Methods: A retrospective database study was conducted among children in ten governmental hospitals in the Tigray region. Gene Xpert MTB/RIF results of sputum/gastric lavage samples from children with presumptive TB from January 2016 to December 2019 were extracted using a data extraction sheet. Data were collected and analyzed using Statistical Package for the Social Sciences version 21.Results: The prevalence of bacteriologically confirmed MTB by Gene-Xpert in children with presumptive TB was 7.3% (95% CI: 6.7%− 7.9%) and the proportion of those that were Gene-Xpert MTB positive who also have rifampicin resistance was 10.9% (95% CI: 8.2– 13.6%). Older children aged 11– 15 years [AOR = 1.76; 95% CI = 1.33– 2.33, p < 0.001] and adolescents 16– 17 years [AOR = 2.18; 95% CI = 1.63– 2.92, p < 0.001] were more likely to be MTB positive. Relapse cases [AOR = 1.66; 95% CI = 1.09– 2.51, p = 0.017] and lost/failure cases [AOR = 8.82; 95% CI = 3.94–