Staphylococci are commensal organisms of animals, but some species are opportunistic pathogens that are resistant to almost all antimicrobial agents in clinical use. Bacterial resistance to β-lactam antimicrobial agents is widespread and has been investigated in species isolated from humans in addition to food production and companion animals. However, minimal progress has been made towards identifying reservoirs of β-lactam-resistant staphylococci in wildlife. This study was aimed at investigating and characterising β-lactamase resistance from staphylococci of wallaby origin. Staphylococci from free-ranging and captive wallabies were assessed for their phenotypic susceptibility to β-lactam antimicrobial agents prior to sequence analysis of their blaZ and blaR1 genes. Deduced amino acid sequences were classified according to the Ambler molecular characterisation method, assigned a protein signature type and compared with sequences generated from previous studies involving isolates from humans, cattle and companion animals. All BlaZ sequences identified in this study were assignable to a pre-existing β-lactamase class and protein signature type, including the more recently discovered protein signature type 12. Three major phylogenetic groups were resolved upon phylogenetic analysis against published BlaZ sequences. This study has found antibiotic-resistant staphylococci both in free-ranging and captive wallaby populations and these bacteria harbour blaZ variants that are different to those recovered from humans, cattle and companion animals. Further studies of staphylococci from non-traditional sources are required in order to enhance our knowledge of the epidemiology of antibiotic resistance genes.