Salmonella enterica subsp. enterica serovar Enteritidis is a major food-borne pathogen that caused most of Salmonella infections worldwide. S. Enteritidis phage type 4 (PT4) especially presents a real challenge for the classical typing methods. We developed a simple multiple-locus variable-number tandem repeat analysis (MLVA) assay based on three hypervariable variable-number tandem repeat (VNTR) loci for subtyping of Salmonella Enteritidis. Testing an arbitrary chosen strain collection of 110 S. Enteritidis isolates, comprising PTs 4, 8, and 21, the MLVA assay yielded a higher discriminatory power, corresponding to a Simpson's index of diversity (ID) of 0.91, when compared to pulsed-field gel electrophoresis (PFGE) which had a Simpson's ID of 0.41. To simplify interpretation of results, we developed a VNTR allele code based on the repeat unit number. This code can easily be exchanged. In conclusion, MLVA is a promising new tool to investigate outbreaks of S. Enteritidis and constitutes a useful addition to the current phage typing scheme.