Salmonella enterica subspecies arizonae (subspecies IIIa) is most frequently associated with reptiles but is also a bacterial pathogen of poultry, primarily of young turkeys where it induces septicemia, neurologic signs, and increased mortality. Arizonosis clinical cases in broiler chickens have recently been documented in the United States, driving the development of a rapid, molecular-based diagnostic for this subspecies. S. enterica subsp. arizonae is a genetically distinct subgroup of S. enterica, primarily diagnosed through culture followed by serotyping or biochemical identification, which are costly in both time and laboratory resources. Real-time/quantitative PCR offers rapid and sensitive detection of Salmonella sp. in laboratory and diagnostic samples; however, no such methodology exists to differentiate S. enterica subsp. arizonae from other Salmonella sp. In this study, we designed a quantitative PCR assay for S. enterica subsp. arizonae. The assay is able to differentiate S. enterica subsp. arizonae from other S. enterica subspecies, including S. enterica subsp. diarizonae (IIIb), and other non-Salmonella bacteria. Validation, including 56 different S. enterica subsp. arizonae serovars, demonstrated 100% sensitivity and 100% specificity. This assay provides a rapid diagnostic option for suspected cases of arizonosis in poultry.