Polymerase chain reaction (PCR) has become an important diagnostic tool in the detection of foodborne pathogens. Many PCR tests have been validated, harmonized, and commercialized to make PCR a standard tool used by food microbiology laboratories to detect pathogens in foods. Current PCR technology allows for rapid detection of pathogens in real time. Real-time PCR can provide qualitative as well as quantitative information. However, PCR does have its limitations because of false-negative and false-positive results that may be encountered with the daily running of PCR assays by a diagnostic laboratory. The intent of this review is to help the reader identify these problems as they occur, discuss the nature of this interference, and provide solutions. This review also discusses the future of molecular diagnostics, i.e., high throughput nucleic acid sequencing.