Nucleotide sequence differences within several virulence genes, including the listeriolysin O (hly) gene, are associated with three evolutionary lineage groups of Listeria monocytogenes. Because the ability of L. monocytogenes to cause disease may vary by evolutionary lineage group, rapid discrimination among the three lineage types may be important for estimating pathogenic potential. A Mismatch Amplification Mutation Assay (MAMA) was developed and used to rapidly screen and characterize L. monocytogenes isolates with regard to lineage type. A standard PCR amplified a 446-bp region within the hly gene with all three L. monocytogenes lineage genotypes. MAMA primers to four different sites within this region of the hly gene were designed to amplify under the same PCR conditions and generated amplicons, the size of which depended on the isolate genotype. Ninety-seven L. monocytogenes isolates were screened. All isolates, except ATCC 19116, could be classified by MAMA PCR as one of the three hly genotypes. Overall, 56, 36, and 4 of the 97 isolates tested were type 1, 2, or 3 respectively. Among the 26 patient isolates, 85%, 15%, and 0% were type 1, 2, or 3 respectively; for the 60 food isolates, 54% were type 1, 43% were type 2, and 3% were type 3. The combination of these MAMA PCR analyses provides a rapid method to screen and categorize L. monocytogenes isolates because of conserved nucleotide differences within the hly gene.