Irish breeder and intensive broiler flocks together with the corresponding poultry farm environment were sampled for the presence of Campylobacter with the aim of identifying potential sources and transmission routes of poultry flock contamination. The genetic diversity of a subset of Campylobacter isolates was examined by analysis of the flaA-short variable region (SVR). Additional discrimination for a further subset of these isolates was achieved using multilocus sequence typing (MLST) analysis. Twenty-four flaA-SVR alleles and 15 FlaA peptides were detected among 92 Campylobacter jejuni and Campylobacter coli isolates. MLST data have been determined for 30 of 92 (32.6%) flaA-SVR-typed isolates with 13 sequence types (STs) present, which were assigned to seven clonal complexes. ST45 was the most common ST identified. Vertical transmission was not found to have played a role in transmission of the pathogen to the poultry flocks. Subtyping by flaA-SVR and MLST identified the practice of partial thinning of flocks as a potential source and route of flock contamination on one broiler farm and implicated a probable source of flock contamination on another. Although there have been several studies reported in the scientific literature, the findings from this study confirmed previous studies and suggested some new transmission pathways including via transport crates. Cross-contamination from adjacent cattle is a new development, and molecular evidence of the role of transport crates in introducing Campylobacter spp. into the broiler house is a recent finding. Further, this study reports the discovery of five new flaA-SVR allele types and eight new STs. These were widespread and persistent in the poultry environment. This new knowledge may explain why despite the on-farm Campylobacter data published to date, there are still no completely effective on-farm control measures to prevent Campylobacter contamination of broiler flocks.