Aims: To characterize horA and its flanking regions of Pediococcus damnosus ABBC478 and, on the basis of this insight, to develop a more specific and sensitive horA PCR method. Methods and Results: A plasmid harbouring the homologue of a hop‐resistance gene, horA, was sequenced and designated pRH478. The nucleotide sequence and open reading frame structure of horA and its flanking regions of pRH478 were found to be highly similar to those of pRH45, a horA‐harbouring plasmid previously identified in Lactobacillus brevis ABBC45. The nucleotide sequence of the horA homologue of P. damnosus ABBC478 was 99·6% identical with that of horA. Based on this insight, new primers specific to horA were designed and compared with the previously reported specific primer pair. As a consequence, it was demonstrated that the new primer pair is superior in specificity and sensitivity. Conclusions: The newly developed horA PCR method allows more specific and sensitive determination of the beer‐spoilage ability of lactic acid bacteria (LAB). Significance and Impact of the Study: The nucleotide sequences of the horA homologues were found to be essentially identical among distinct species of LAB, indicating that horA‐specific primers can be designed from almost any region of the horA gene.