A novel real-time PCR assay was developed for the direct detection in food of Helicobacter pullorum-like bacteria, which are occasionally associated with human enteric disease. Experiments using control strains showed that the realtime PCR assay was specific and reproducible, with a detection level of 1 colony-forming unit (CFU)/g. The assay was then applied to determine contamination rates in 30 samples of three types of chicken-meat products obtained from five retail outlets in Spain (Valencia); all of the samples were initially considered to be culture-negative for Helicobacter even after an enrichment period. H.pullorum-like DNA was detected in seven out of ten chicken carcasses and in one chicken-burger sample (without enrichment), as well as in one liver sample (after enrichment). Sequencing of three randomly selected PCR products confirmed concordance (99% homology) with the H. pullorum 16S rDNA gene. The advantages of real-time PCR over conventional PCR assays are the improved detection level, speed of testing, and validation of specificity by melting-point analysis. The fact that bacteria are frequently present in chicken carcasses sold in retail stores highlights the importance of more widely monitoring contamination rates. The novel assay described herein allows better assessment of potential human health risks posed by H. pullorum.