Background <p>Rifampicin-resistant tuberculosis (RR-TB) remains a public health challenge, with suboptimal treatment outcomes and challenging management of contacts. Whole genome sequencing (WGS) generates a comprehensive drug resistance profile and transmission inference with a single assay. Limited evidence exists on how healthcare workers (HCWs) perceive the value of WGS, especially for post-exposure management.</p> Methods <p>We conducted a qualitative study with 22 purposively sampled HCWs (9 doctors, 9 nurses, 4 community health workers) at 11 public health care facilities in the Western Cape, South Africa. Following a standardized explanation of WGS and WGS-guided post-exposure management, semi-structured interviews were conducted to elicit responses on anticipated acceptability and value of WGS for post-exposure management. Interviews were audio-recorded, transcribed, and analysed using thematic analysis, combining inductive with deductive approaches informed by the Theoretical Framework of Acceptability themes of intervention coherence, self-efficacy, affective attitude, ethicality, perceived effectiveness and opportunity costs.</p> Results <p>HCWs demonstrated good anticipated intervention coherence, even though nurses and community health workers required more explanation on WGS than doctors, and pathways for WGS integration into routine care were not clear. Most HCWs indicated high confidence in anticipated application of WGS-supported recommendations. Limited familiarity with genomic tools was identified as a barrier that requires formal training. Affective attitudes were largely positive, with HCWs expressing optimism about improved clinical decision-making and reduced diagnostic uncertainty following access to WGS. Anticipated effectiveness was high, with HCWs hopeful of faster initiation of appropriate regimens for patients and contacts, reduced patient distress, and decreased medication wastage. Less applicable to post-exposure management was the anticipated benefit that WGS could reduce the burden to the health system and patients by limiting repeat visits and reducing laboratory delays. Ethicality emerged as an important advantage, with HCWs anticipating reduced stigma when contact screening is guided by the detection of hotspots. HCWs did not anticipate opportunity costs.</p> Conclusion <p>HCWs in high-burden settings generally anticipated that WGS would be acceptable to inform post-exposure management strategies. Successful integration of WGS into routine TB care will require tailored training and clear implementation guidance on how to interpret and apply WGS results in clinical and public health practice.</p> Clinical trial number <p>Not applicable.</p>