Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius are thermo-acidophilic, non-pathogenic, spore-forming bacteria that can survive the typical heat processing of fruit juices and concentrates. Bacterial endospores then germinate, grow and cause spoilage of acid food products. Species of Alicyclobacillus were isolated from orchard soil and a fruit concentrate production factory in South Africa. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was done by PCR amplification using genus-specific primers and 16S ribosomal RNA (rRNA) gene sequencing. The majority of isolates belonged to the species A. acidoterrestris, but A. acidocaldarius was also isolated and identified. As far as we could determine, this is the first report of the isolation of A. acidoterrestris from wash water and soil outside a fruit processing plant, as well as the isolation of A. acidocaldarius from vinigar flies. The genotypic relatedness between strains of A. acidoterrestris and between strains of A. acidocaldarius was determined by RAPD-PCR. Sixteen isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Three isolateT:/PGN/ELSEVIER/YFMIC/web/00001155/s identified as A. acidocaldarius gave three unique banding patterns.