Listeria monocytogenes has been implicated in a number of outbreaks involving fresh produce, including an outbreak in 2016 resulting from contaminated packaged salads. The persistence and internalization potential of L. monocytogenes in romaine lettuce was evaluated, and the persistence of two L. monocytogenes strains was assessed on three romaine lettuce cultivars. Seeds were germinated, and plants grown in three soil types (i.e., standard potting mix, autoclaved potting mix, and top soil) and sterile soft-top agar for up to 21 days. Average CFU per gram of L. monocytogenes on seeds and plants was calculated from five replicates per harvest day. Up to 8.2 log CFU/g L. monocytogenes persisted on romaine lettuce plants (Braveheart cultivar) grown in soft-top agar, while those grown in commercial potting mix (initial soil aerobic plate count of 4.0 × 104 CFU/g) had a final concentration of 5.4 log CFU/g, and autoclaved commercial potting mix had a final concentration of 3.8 ± 0.2 log CFU/g after a 21-day period. Pathogen levels dropped below the limit of detection (2 log CFU/g) by day 18 in 75% topsoil (initial soil aerobic plate count of 4.0 × 101 CFU/g); this did not occur in sterile media. Although L. monocytogenes strain differences and presence of a clay coating on seeds did not affect persistence, differences were observed in L. monocytogenes growth and survival among cultivars. To assess internalization, seeds were inoculated with L. monocytogenes expressing green fluorescent protein. Three plants were fixed, paraffin embedded, and sectioned; localization was studied by using standard immunohistochemistry techniques. A total of 539 internalized L. monocytogenes cells were visualized among three 20-day seedlings. L. monocytogenes cells were located in all major tissue types (pith followed by cortex, xylem, phloem, and epidermis). The presence of L. monocytogenes in the plant vasculature suggests potential for transport throughout the plant into edible tissue.