OBJECTIVES: High-level β-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of β-lactam resistance and sought to understand the molecular basis of the resistance observed. METHODS: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the β-lactam resistance profiles for the isolates and the isolates were further investigated by WGS. RESULTS: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. CONCLUSIONS: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased β-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.