Two novel methods that allow the powerful identification of Listeria monocytogenes by polymerase chain reaction (PCR) and simultaneous differentiation by special electrophoresis formats are described. The first method involves a PCR-driven single-strand conformation polymorphism (SSCP-PCR) assay using a portion of the noncoding region of the hlv gene. The assay was evaluated with 120 genetically distinct L. monocytogenes strains of either foodborne or clinical origin. Distribution of listerial strains to at least 14 SSCP types was observed. In respect to the panel of strains, 39.7% were assigned to SSCP type 3, and 19% showed SSCP type 5. Further, SSCP type 1 was found in 7.5% of all strains, SSCP type 10 in 6.7%, and 5.8% each for SSCP types 6 and 7. The SSCP types 4, 9, and 11 were infrequently described in 2.55%, 3.3%, and 4.2%, respectively, of all isolates. At least 0.85% represented each of the SSCP types 2, 13, and 14, and 1.7% displayed SSCP types 8 and 12. In the second method, the internal threonine-asparagine repeat portion of the L. monocytogenes p60 protein was used for setting up a PCR-based identification and parallel differentiation assay. Ten different repeat types (RTs), according to different sizes of PCR products, were observed. Of 163 strains tested, 35.58% of samples were assigned to RT 1, 39.26% to RT 2, 3.68% to RT 3, 6.13% to RT 4, 4.29% to RT 5, 2.45% to RT 6, 5.52% to RT 7, 0.61% to RT 8, 0.61% to RT 9, and 1.83% to RT 10. The data suggest that both methods allow the simple identification and differentiation of L. monocytogenes isolates. Therefore, both the SSCP-PCR and the PCR-based identification and parallel differentiation assay could represent single-strand pretyping assays before laborious reference typing methods are applied.