BACKGROUND: In the absence of reliable data on drug-resistant TB in Eritrea, a national survey was conducted in 2018 using molecular-based methods, bypassing the need for culture.METHODS: A cross-sectional study was conducted in all 77 TB microscopy centres in the country. All 629 newly registered sputum smear-positive pulmonary TB patients were enrolled over 12 months. Sputum samples were tested using the Xpert® MTB/RIF assay and targeted next-generation sequencing (Deeplex Myc-TB) to identify resistance and explore the phylogenetics of Mycobacterium tuberculosis complex strains.RESULTS: Drug resistance profiles were obtained for 555 patients (502 new, 53 previously treated). The prevalence of rifampicin-resistant TB (RR-TB) was respectively 2.0% and 7.6% among new and previously treated cases. All RR-TB isolates that were susceptible to isoniazid displayed a phylogenetic marker conferring capreomycin resistance, confirming circulation of a previously described resistant TB sub-lineage in the Horn of Africa. Only one case of fluoroquinolone resistance was detected.CONCLUSION: The prevalence of rifampicin resistance among TB patients is encouragingly low. The scarcity of fluoroquinolone resistance bodes well for the success of the recommended all-oral treatment regimen. Surveillance based on molecular approaches enables a reliable estimation of the burden of resistance and can be used to guide appropriate treatment and care.