Technical Abstract: Listeria monocytgenes is a significant food-borne human and veterinary pathogen. Poor quality silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis cases that occurred on two Pacific Northwest dairy farms revealed that the bovine listeriosis strains from both farms were closely related when subtyped using pulsed-field gel electrophoresis (PFGE). Additionally, the bovine strains were identical or closely related to silage and fecal strains present on the farms. One of the farms was heavily contaminated with L. monocytogenes and environmental samples were collected from this farm at two time points over nine months. PFGE characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars present. Fecal isolates obtained from individual cows were the most genetically diverse with up to 94% of fecal samples containing more than one pulsovar. The maximum number of pulsovars and serotypes isolated from the fecal sample of one cow was 6 and 4, respectively. Serotype 1/2a strains were isolated most frequently at both time points and were more persistent as compared to other serotypes. Persistent and nonpersistent strains were assayed for biofilm formation and all strains were capable of biofilm formation, although the quantity of biofilm formation was more variable for nonpersistent strains. Microarray analysis of bovine listeriosis strains and closely related strains from feces and silage identified several probes that differentiated listeriosis strains from environmental strains, however no probe was identified as characteristic of both listeriosis strains.