Putative routes of Listeria monocytogenes contamination, based on the workflow of the employees, were studied in a meat processing facility by investigating 226 samples collected from food contact surfaces, non-food contact surfaces, raw materials, and ready-to-eat meat products on four occasions over a 1-year period. In total, 19.7% of non-food contact surfaces, 22.9% of food contact surfaces, 45% of raw materials, and 20% of ready-to-eat meat products were positive for L. monocytogenes (analyzed by the International Organization for Standardization standard method ISO 11290). Pulsed-field gel electrophoresis (PFGE) profiles were determined for a representative subset of these isolates, and 11 distinct pulsotypes were identified, two of which were frequently isolated (T4 and T8) and considered persistent. Strains from the various pulsotypes were screened for the presence of bcrABC and qacH, the genes responsible for tolerance responses to quaternary ammonium compounds. Two strains harbored bcrABC, and these strains had a higher benzalkonium chloride tolerance; however, they were not considered persistent strains. The frequently isolated PFGE pulsotype T8 strains were highly adhesive to abiotic surfaces at 10 and 20°C; however, the pulsotype T6 strain, which was isolated only at the last sampling time, had the highest adhesion ability, and the pulsotype T4 strain (the second most persistent pulsotype) had only modest adhesion. Four putative cross-contamination routes were confirmed by mapping the persistent and other isolates. This information could allow a food safety manager to adjust the work flow to improve the hygienic conditions in a meat processing facility. This study revealed the prevalence and persistence of L. monocytogenes strains in a meat processing facility and established the importance of developing strategies to avoid cross-contamination, recalls, and outbreaks of listeriosis.