Some Alicyclobacillus species cause the spoilage of acidic beverages resulting in an odor of phenolic compounds such as guaiacol. We have already developed a discrimination method based on the gyrB sequence for A. acidoterrestris , which is the most frequently isolated species from spoiled products. Furthermore, in order to discriminate the other Alicyclobacillus species, we determined gyrB sequences of additional Alicyclobacillus species and comparative analysis of gyrB sequences was performed among all Alicyclobacillus species. The results showed species-specific regions in each Alicyclobacillus species. Using these regions as primers, each Alicyclobacillus species could be clearly detected with the PCR technique. Moreover these primer sequences could be applied to the LAMP method, and each Alicyclobacillus species could be rapidly and easily detected without amplification of non-target species. It is suggested that these species-specific regions are useful for discrimination of Alicyclobacillus at species level in quality control laboratories.