Contamination of fresh produce with Listeria monocytogenes has resulted in outbreaks of systemic listeriosis and febrile gastroenteritis. Recalls of alfalfa sprouts have occurred due to contamination with L. monocytogenes. Alfalfa sprouts were used as a preharvest model to study the interaction with this human pathogen. Seventeen strains were assessed for their capacity to colonize alfalfa sprouts, and strain-specific differences (not related to source, serotype, or lineage) were revealed when the sprout irrigation water was changed daily. Two of the strains colonized and attached to the sprouts very well, reaching levels of more than 5 log CFU per sprout. The remaining strains varied in their final levels on sprouts between less than 1 to 4.7 log CFU per sprout. All of the L. monocytogenes strains grew to equivalent levels on the sprouts when the irrigation water was not changed, suggesting the differences observed with regular changing of the water resulted from differences in attachment. Further analysis of the best colonizing strains indicated that only between 0.3 and 1 log CFU per sprout could be removed by additional washing of the sprout, and the presence of normal sprout bacteria did not compete with the L. monocytogenes strains on the sprouts. The poorest colonizing strain was able to grow in the irrigation water during the experiment but could not attach to the sprouts. Microscopic examination of the sprouts with L. monocytogenes expressing the green fluorescent protein indicated that L. monocytogenes was associated with the root hairs of the sprouting alfalfa, with few to no cells visible elsewhere on the sprout.