The objective of the present investigation was to differentiate between strains of Streptococcus equi subspecies equi implicated in abscess formation in vaccinated horses. Streptococcus equi isolates recovered from clinical specimens associated with equine strangles cases submitted to the University of Illinois Veterinary Diagnostic Laboratory were compared with S. equi isolates representing at least 12 lots of a commercial modified live vaccine (MLV) to determine whether the isolates obtained from the abscesses were vaccine or wild type. Genotyping techniques evaluated included enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), repetitive extragenic palindrome PCR, BOX element PCR, ribotyping, and pulsed-field gel electrophoresis (PFGE). Phenotypic evaluations were performed using the Biolog GP2 Microplate (hereafter, Biolog). In cases where Biolog and PFGE results did not coincide, a single nucleotide polymorphism located in the upstream regulatory region of szp gene was used to identify the S. equi strains. PFGE and Biolog successfully differentiated wild-type S. equi strains isolated from clinical submissions from isolates of the MLV. PFGE genotyping enabled further subtyping of the wild-type strains, whereas Biolog combined with szp sequencing was useful in differentiating the MLV strain from its wild-type progenitor. Deletion of a single guanine residue located in the upstream regulatory region of the szp gene appears to be conserved among vaccine isolates, and shows a 98.5% correlation to Biolog identification. This multiphasic approach can be used to answer specific diagnostic questions pertaining to the source of infection and/or outbreak, or to address quarantine concerns.