In an attempt to study the diversity and persistence of molecular subtypes of pathogenic Listeria spp. in a cheese factory at the La Mancha region of Spain, 43 samples were taken from incoming raw milk (cow's, ewe's, goat's and mixed species) and from certain food-contact and environmental surfaces before and/or after sanitation. Of these samples, 12 contained pathogenic Listeria. From the chromogenic agar plates corresponding to those, 46 phosphatidylinositol-specific phospholipase C-positive isolates were randomly taken for further analysis, including biochemical tests and pulsed-field gel electrophoresis (PFGE). They coincided in identifying all the 46 as Listeria ivanovii subsp. ivanovii, apparently a single PFGE type. Both ewe's and goat's raw milk batches from asymptomatic animals tested along the 6-month period persistently carried the same strain, which was also obtained from inner surfaces of raw milk truck tanks and the milk dump tank at the cheese factory. Biofilm-forming abilities of this L. ivanovii clone and interference against L. monocytogenes Scott A reference strain were tested, but failed to account for the clone's apparent pervasive presence.