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BackgroundCollecting data on antimicrobial resistance (AMR) is an essential approach for defining the scope of the AMR problem, developing evidence-based interventions and detecting new and emerging resistances. Our study aimed to identify key factors influencing the implementation of a laboratory-based AMR surveillance system in Cambodia. This will add additional insights to the development of a sustainable and effective national AMR surveillance system in Cambodia and other low- and middle-income countries.MethodsKey informants with a role in governing or contributing data to the laboratory-based surveillance system were interviewed. Emerging themes were identified using the framework analysis method. Laboratories contributing to the AMR surveillance system were assessed on their capacity to conduct quality testing and report data. The laboratory assessment tool (LAT), developed by the World Health Organisation (WHO), was adapted for assessment of a diagnostic microbiology laboratory covering quality management, financial and human resources, data management, microbiology testing performance and surveillance capacity.ResultsKey informants identified inadequate access to laboratory supplies, an unsustainable financing system, limited capacity to collect representative data and a weak workforce to be the main barriers to implementing an effective surveillance system. Consistent engagement between microbiology staff and clinicians were reported to be a key factor in generating more representative data for the surveillance system. The laboratory assessments identified issues with quality assurance and data analysis which may reduce the quality of data being sent to the surveillance system and limit the facility-level utilisation of aggregated data. A weak surveillance network and poor guidance for outbreak response were also identified, which can reduce the laboratories’ opportunities in detecting critical or emerging resistance occurring in the community or outside of the hospital’s geographical coverage.ConclusionThis study identified two primary concerns: ensuring a sustainable and quality functioning of microbiology services at public healthcare facilities and overcoming sampling bias at sentinel sites. These issues hinder Cambodia’s national AMR surveillance system from generating reliable evidence to incorporate into public health measures or clinical interventions. These findings suggest that more investments need to be made into microbiology diagnostics and to reform current surveillance strategies for enhanced sampling of AMR cases at hospitals.