ABSTRACT Listeria monocytogenes can survive in food production facilities and can be transmitted via contamination of food during the various stages of food production. This study was conducted to compile the results of three independent previous studies on the genetic diversity of L. monocytogenes in a poultry production company in Spain and to determine the potential virulence and sanitizer resistance of the strains by using both genotype and phenotype analyses. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain. A set of 347 L. monocytogenes isolates representing 39 subtypes was obtained using pulsed-field gel electrophoresis (PFGE). A total of 28 subtypes (68%) had a full-length internalin A gene, and two subtypes had a phenotype with low potential for virulence because of a mutation in the prfA gene. A total of 32 subtypes (82%) were classified as benzalkonium chloride resistant (BAC-R) and contained the resistance determinant bcrABC (21 subtypes, 54%) or the resistance gene qacH (11 subtypes, 28%). A total of 13 persistent BAC-R subtypes (minimum of 3 months between the first and last sample from with the isolate was recovered) were identified at the abattoir and processing plant. The three production stages shared a unique subtype (PFGE type 1), which had the mutation in the prfA gene and the bcrABC resistance determinant. Whole genome sequencing revealed this subtype to be sequence type 31. Limited genetic diversity was noted in the isolates studied, including some subtypes that were persistent in the environment of the investigated facilities. Given the high prevalence of BAC-R subtypes, these results support the association between resistance to biocides and persistence of L. monocytogenes . HIGHLIGHTS Subtyping of L. monocytogenes isolates in poultry production revealed limited diversity. A set of 347 isolates consisted of 39 individual subtypes. A total of 28 subtypes had a full-length internalin A gene. A total of 32 subtypes were classified as benzalkonium chloride resistant. The study results support the association between resistance to biocides and persistence.