Abstract. Background. Prosthetic joint infection (PJI) is a significant cause of morbidity and mortality following knee replacement surgery. Identifying the causative agent(s) and their antibiotic sensitivities is critical in determining the choice of treatment methods used and the likelihood of successful eradication. This study aimed to investigate:. Whether biopsy alone was superior to aspiration alone in specificity and sensitivity for diagnosing PJI following knee replacement. Whether biopsy identifies the same microbiological flora as aspiration. Methods. We identified consecutive patients passing through our knee infection Multi-Disciplinary Team meeting between December 2014 and March 2020. Data was collated data retrospectively using electronic records. Statistical analysis was performed using Stata (Timberlake, February 2020). Results. 65 of 100 patients identified had both pre-operative aspiration and biopsy. In 29% of positive patients, biopsy identified new or additional organisms not previously identified by aspiration. Aspiration had a sensitivity of 70%, specificity of 88%, positive predictive value of 90.3% and negative predictive value of 64.7%. Biopsy had a sensitivity of 97.5%, specificity of 88%, positive predictive value of 92.9% and negative predictive value of 95.7%. Conclusion. Biopsy is superior to aspiration in the diagnosis of PJI and can be performed safely and successfully. It identifies organisms when aspiration may be negative and identifies additional microorganisms in a polymicrobial setting not identified by aspiration alone (29% of positive cases). We would recommend, where possible, aspiration and biopsy as routine pre-operative sampling in order to identify all causative agents and their susceptibilities prior to embarking on revision surgery