Alicyclobacillus spp. are a group of thermophilic, acidophilic, spore-forming bacteria, some of which cause spoilage in commercially pasteurized fruit and vegetable juice products including apple, orange, tomato and carrot juice. In this study, we characterized seven isolates of Alicyclobacillus (14-2, KF, A-Gala 2-1, C-Fuji 6, Gala 9-2, 1016, 18-1) and a reference strain A. acidiphilus DSM 14558 by a 16S rDNA sequencing technique and Fourier transform infrared (FT-IR) spectral features. The degree of similarity between Alicyclobacillus isolates based upon phylogenetic analysis of sequence data and multivariate statistical analysis of FT-IR spectral data was determined. Results from both methods were consistent, suggesting that a combination of methods targeting both 16S rDNA and mid-infrared spectroscopic signatures may be a suitable and more accurate approach to either identify or discriminate Alicyclobacillus isolates from fruit and vegetable juice products.