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SUMMARY: In viral genomic research and surveillance, inter-sample contamination can affect variant detection, analysis of within-host evolution, outbreak reconstruction, and detection of superinfections and recombination events. While sample barcoding methods exist to track inter-sample contamination, they are not always used and can only detect contamination in the experimental pipeline from the point they are added. The underlying genomic information in a sample, however, carries information about inter-sample contamination occurring at any stage. Here, we present Polyphonia, a tool for detecting inter-sample contamination directly from deep sequencing data without the need for additional controls, using intrahost variant frequencies. We apply Polyphonia to 1 102 SARS-CoV-2 samples sequenced at the Ó³»­´«Ã½ and already tracked using molecular barcoding for comparison.AVAILABILITY AND IMPLEMENTATION: Polyphonia is available as a standalone Docker image and is also included as part of viral-ngs, available in Dockstore. Full documentation, source code, and instructions for use are available at .SUPPLEMENTARY INFORMATION: Data for reproducing results are available at Bioinformatics online.