Background: Tuberculosis is an infectious disease usually caused by Mycobacterium tuberculosis bacteria. The global emergence of mono- or multidrug-resistant tuberculosis and extensively drug-resistant forms of tuberculosis poses a considerable challenge to tuberculosis control programs. There has been no reliable and organized data on trends and drug resistance of Mycobacterium tuberculosis in the study area. Therefore, this study aimed to determine the trends of Mycobacterium tuberculosis and Rifampicin resistance in the Adigrat General Hospital, eastern zone, Tigray, Northern Ethiopia.Methods: A hospital-based retrospective cross-sectional study was conducted at Adigrat General Hospital from January 2015 to 2018. Data were collected retrospectively from the GenexpertTM TB registration books using a data extraction format. Data was entered into Epi-Info 3.1 and subsequently exported and analyzed using SPSS Version 20. The results were summarized using descriptive statistics, tables, and figures. P values < 0.05 were considered statistically significant.Result: A total of 5,944 Mycobacterium tuberculosis presumptive patients were included in the study. The majority of the study participants were male (58.1%) with participants’ median age of 40.0 (IQR 57, 26) years, the majority were 30-44 years. The overall positive cases of Mycobacterium tuberculosis was 24.3% (1446) with a total of 132 (9.1%) found to be resistant to rifampicin. Within the total Rifampicin resistant sub-group, 129/132 (97.7 %) were newly identified cases and the rest were previously treated, tuberculosis patients. Age, the reason for diagnosis, site of presumptive tuberculosis, and/or being HIV infected showed significant association with our dependent variable; however, only age and being HIV infected were associated with rifampicin resistance.Conclusion: In our study, the overall trends of Mycobacterium tuberculosis and rifampicin resistance were found to be high and gradually increasing. HIV co-infected and previously treated patients were more likely to develop rifampicin resistance. Therefore, maximizing early detection of drug-resistant Mycobacterium tuberculosis and strengthening tuberculosis infection control activities are recommended to reduce the burden of this contagious and potentially deadly disease.