Currently the primary method for confirming acute SARS-CoV-2 infection is through the use of molecular assays that target highly conserved regions within the viral genome . Many, if not most of the diagnostic targets currently in use were produced early in the pandemic, using genomes sequenced and shared in early 2020 . As viral diversity increases, mutations may arise in diagnostic target sites that have an impact on the performance of diagnostic tests . Here, we report on a local outbreak of SARS-CoV-2 which had gained an additional mutation at position 28890 of the nucleocapsid protein, on a background of pre-existing mutations at positions 28881 , 28882 , 28883 in one of the main circulating viral lineages in Wales at that time . The impact of this additional mutation had a statistically significant impact on the Ct value reported for the N gene target designed by the Chinese CDC and used in a number of commercial diagnostic products . Further investigation identified that, in viral genomes sequenced from Wales over the summer of 2020, the N gene had a higher rate of mutations in diagnostic target sites than other targets, with 115 issues identified affecting over 10% of all cases sequenced between February and the end of August 2020 . In comparison an issue was identified for ORFab, the next most affected target, in less than 1.4% of cases over the same time period . This work emphasises the potential impact that mutations in diagnostic target sites can have on tracking local outbreaks, as well as demonstrating the value of genomics as a routine tool for identifying and explaining potential diagnostic primer issues as part of a laboratory quality management system . This work also indicates that with increasing genomic sequencing data availability, there is a need to re-evaluate the diagnostic targets that are in use for SARS-CoV-2 testing, to better target regions that are now demonstrated to be of lower variability.