Lactobacillus wasatchensis is a slow-growing non-starter lactic acid bacterium (NSLAB) recently implicated in gassy defects in aged Cheddar cheese. This organism has been detected in cheeses from 7 cheese processing facilities in different regions of the United States and is of significant concern to cheese producers. Rapid detection of Lb. wasatchensis would allow for better control of the organism, and help determine where it is entering the manufacturing process. A set of 16S rRNA primers were developed using NCBI Primer-Blast against the Lb. wasatchensis genome and selected based on product length, melting temperature, and primer self-complementarity. In silico analysis against the NCBI database indicated the primers should have high specificity for Lb. wasatchensis. PCR optimum conditions were determined experimentally with Lactobacillus casei and Lactobacillus curvatus DNA as non-target template. To determine specificity, the primers were tested against DNA extracted from 22 different common NSLAB, including strains of Lb. wasatchensis isolated from cheese and the original Lb. wasatchensis WDC04. Only strains identified previously as Lb. wasatchensis amplified with the primers. Even the mostly closely related NSLAB species (such as Lb. curvatus) to Lb. wasatchensis could be differentiated with these primers. DNA from all isolates amplified using standard bacterial 16S rRNA primers. The new primers, LW86Fa and LW258Ra, will be used in traditional and real-time PCR for rapid detection of Lb. wasatchensis in gassy cheeses and the cheese processing environment. Rapid molecular detection will help diagnose and track Lb. wasatchensis contamination, and help control the occurrence of gassy-cheese defects.