Mycobacterial infections cause a reasonable burden of morbidity and mortality in global feline populations, many of which are ‘Vulnerable’ or ‘Endangered’. Identifying these infections may facilitate efforts to protect these animals. An interferon-gamma (IFNγ) release assay (IGRA) to diagnose mycobacteriosis in domestic cats has been adapted for use in lions; however, the development of species-specific antibodies may be laborious. Therefore, we investigated whether anti-cat IFNγ antibodies can bind to recombinant IFNγ (rIFNγ) from other Felidae species, permitting use of the feline IGRA in a wider range of felids. Unique Felidae IFNγ protein sequences and their corresponding coding nucleotide sequence were identified from online databases; plasmids with an IFNγ-gene insert were synthesised to transform E. coli-DH5α and subsequently transfect HEK 293T cells to secrete rIFNγ. Enzyme-linked immunosorbent assay using a commercial anti-cat IFNγ kit was performed to detect rIFNγ from Felidae, the domestic dog and cattle. Five unique rIFNγ Felidae proteins were synthesised; anti-cat IFNγ antibodies were able to bind to all five proteins, while cross-reactivity with canine and bovine rIFNγ was negligible. This suggests that anti-cat IFNγ antibodies are sufficient for detection of IFNγ across other Felidae species, namely the lion, tiger, cheetah, cougar, Iberian lynx and the Canadian lynx.