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A real-time polymerase chain reaction (PCR) assay, amplifying the genes encoding lactose permease (lacY) and invasion plasmid antigen H (ipaH), was run on 121 isolates phenotypically classified as Shigella spp., enteroinvasive Escherichia coli (EIEC), or EIEC O nontypable (ONT). The results were compared with data from a generic E. coli multiple-locus variable-number of tandem repeat analysis (MLVA) and a Shigella MLVA. The real-time PCR verified all Shigella spp. (n = 53) as Shigella (lacY negative) and all EIEC O121 (n = 15) and EIEC O124 (n = 2) as EIEC (lacY positive). However, the real-time PCR typed EIEC O164 as either EIEC (n = 2) or Shigella (n = 2) and, thus, was not suited for classifying this group of isolates. Interestingly, the majority (42/47, 89.4%) of the EIEC ONT were classified as Shigella (lacY negative) by the real-time PCR, and in nearly all cases, (92.9%, 39/42) data from both MLVA assays supported these findings. Overall, in 94.7% (114/121) of the isolates, the results from the real-time PCR were substantiated by the results from the MLVA assays. In conclusion, the real-time PCR assay was fast and accurate in differentiating Shigella spp. from EIEC, with the exception of the EIEC O164 group. This molecular assay was particularly pragmatic for the challenging EIEC ONT group.