A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P<0.05) and samples from other sources (P<0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P<0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.