Many foods originate on the farm where cross-contamination with pathogens can occur, with implications for human health. This study characterized a bank of 51 Listeria monocytogenes isolates originating from 12 farms located in Ireland by pulsed-field gel electrophoresis (PFGE) to establish the molecular diversity of the isolate collection, and examine transmission patterns of L. monocytogenes across the farm environment, and also determined resistances against five different antibiotics (ampicillin, ciprofloxacin, erythromycin, penicillin G, and tetracycline). Analysis using a combination of AscI and ApaI digestion showed the 51 isolates comprised a total of 40 individual PFGE types, compared to individual restriction enzyme analysis, which was less discriminatory (36 types with ApaI analysis and 38 types with AscI analysis). Four of the PFGE types were common to multiple farms, and five farms had isolates with indistinguishable PFGE types in multiple locations on the farm. Indistinguishable PFGE types were common to multiple farms in different geographical locations up to ∼200 km apart, and were found in a variety of different sample types, indicating multiple niches for the organism in the dairy farm environment. The presence of L. monocytogenes in samples related to animals other than cattle indicated that there are multiple possible vectors of contamination. The farm environment harbors a diverse collection of L. monocytogenes isolates that must be considered as possible agents of food contamination.