BGC is a venereal disease characterised by infertility and sporadic abortion, caused by Campylobacter fetus subspecies venerealis. Gold standard diagnosis relies upon culture isolation and differentiation from C. fetus subsp. fetus which can be difficult. Our aim is to improve diagnostic methods for BGC. Fifty-one Campylobacter-like (CL) isolates from bovine prepuces were screened using standard OIE and novel culture protocols, and PCR methods based on ParA and the insertion element ISCfe1. All 51 isolates were positive on one or more PCR assays and 29 were confirmed as C. fetus subsp. venerealis biochemically. Illumina genome sequencing of two Australian strains of C. fetus subsp. venerealis identified 26 conserved specific regions which did not distinguish the subspecies using PCR. Most of these regions appear to be associated with pathogenicity islands or putative virulence genes which could be present in other closely related species. Culture isolation was improved through the addition of filters to exclude contaminants, and the Thomann Transport Medium is more effective compared to current OIE protocols. Culture may remain as the gold standard and molecular tools may be useful for pathogenicity studies. Future pangenomic analyses of several C. fetus subspecies may assist to identify robust diagnostic targets.