Characterization of 80 Listeria monocytogenes isolates from urban and natural environments differentiated 7 and 26 EcoR1 ribotypes, respectively. Whereas the majority of isolates from the natural environment represented L. monocytogenes lineage II (12 of 13 isolates), urban isolates grouped evenly into lineages I and II (32 and 33 isolates, respectively) and included two lineage III isolates. Multilocus sequence typing of all natural isolates and a randomly selected subset of 30 urban isolates showed a higher overall diversity (Simpson index of discrimination [D] of 0.987 and 0.920. respectively) than did EcoR1 ribotyping (D = 0.872 and 0.911, respectively). Combined analysis with ribotype and lineage data for 414 isolates from farm sources, 165 isolates from foods and food-processing environments, and 342 human clinical isolates revealed that lineage I was significantly more common among human (P < 0.0001) isolates, whereas lineage II was more common among isolates from the natural environment, farms, and foods (P ≤ 0.05). Among a total of 92 ribotypes, 31 showed significant associations with specific isolate sources. One ribotype (DUP-1039C) was significantly associated with both natural environments and farms. A spatial analysis showed a marginal association between locations in the natural environment positive for L. monocytogenes and a proximity to farms. Our data indicate that (i) L. monocytogenes strains from different sources show a high level of diversity; (ii) L. monocytogenes subtypes differ significantly in their associations with different environments, even though populations overlap; and (iii) a higher proportion of isolates from environmental sources than from human clinical cases can be classified into L. monocytogenes lineage II, which supports the classification of this lineage as an environmentally adapted subgroup.