Technical Abstract: Salmonella enterica is a prevalent food-borne pathogen that is often associated with poultry. Identification of Salmonella serotype is a required subtyping step in classification of this bacterium. Serotyping is a slow and difficult process. Therefore a molecular method was developed for the rapid and easy determination of Salmonella serotypes. Analysis by comparative genomic hybridization (CGH) determined that each Salmonella enterica subspecies I serotype contains a core of about 4000 genes and an additional 200-600 genes that are serotype specific. Unique genes identified in this work were selected based on their differential distribution among clinically prevalent serovars. A multiplex polymerase chain reaction (PCR) with fluorescently labeled primers was developed to detect these genes by analysis with an automated DNA sequencer. The assay was capable of correctly identifying the top 50 clinical Salmonella serotypes in a rapid high-throughput analysis. The assay has been named SMART for “Salmonella multiplex assay for rapid typing.” SMART is faster, automated, requires less labor, and is cheaper than traditional seroytping ($1.48/isolate versus ~40.00/isolate). This new method enables scientists, clinicians, inspectors, and epidemiologists to rapidly and easily determine Salmonella serotype, an important step in surveillance, treatment, compliance, and outbreak investigation. Implementation of SMART will help improve human health and food-safety.