We used molecular subtyping to investigate an outbreak of listeriosis involving residents of 24 US states. We defined a case as infection with Listeria monocytogenes serotype 4b yielding one of several closely related patterns when subtyped by pulsed-field gel electrophoresis. Patients infected with strains yielding different patterns were used as controls. A total of 108 cases were identified with 14 associated deaths and four miscarriages or stillbirths. A case-control study implicated meat frankfurters as the likely source of infection (OR 17.3, 95% CI 2.4-160). The outbreak ended abruptly following a manufacturer-issued recall, and the outbreak strain was later detected in low levels in the recalled product. A second strain was recovered at higher levels but was not associated with human illness. Our findings suggest that L. monocytogenes strains vary widely in virulence and confirm that large outbreaks can occur even when only low levels of contamination are detected in sampled food. Standardized molecular subtyping and coordinated, multi-jurisdiction investigations can greatly facilitate detection and control of listeriosis outbreaks.