<p><i>Cronobacter sakazakii</i> is an emerging opportunistic food-borne pathogen associated with a wide range of foods including seafood, meat, plant-based, and dairy products. Although seafood is a significant source of this pathogen, specific and sensitive methods that can differentiate <i>C. sakazakii</i> from other <i>Cronobacter</i> species are currently not available. In this study, a PCR assay using primers targeting <i>fhuF</i> gene was developed and evaluated for its specificity and sensitivity in detecting <i>C</i>. <i>sakazakii</i>. The designed primers exhibited high level of specificity for <i>C. sakazakii</i> and no cross reaction was observed with other <i>Cronobacter</i> species and related Enterobacterales. The sensitivity of detection was tested using pure culture of <i>C. sakazakii</i> and in shrimp samples artificially spiked with <i>C. sakazakii</i>. The <i>fhuF</i>-specific PCR assay could detect as low as 1.45 × 10³ cfu/ml in pure bacterial cultures and 2.9&#xa0;cfu/g in spiked shrimp following four hours of enrichment. When applied to natural samples of fresh and dried seafood, the assay detected <i>C. sakazakii</i> in three samples following enrichment in a selective broth. The novel <i>fhuF</i>-specific PCR assay developed in this study provides a reliable and sensitive tool for the detection of <i>C. sakazakii</i> and will be useful in prevalence and risk assessment studies involving seafood.</p>