The genus Alicyclobacillus represents a group of mostly acidophilic, meso-thermophilic bacteria, which obtained their name through the biosynthesis of uncommon, alicyclic fatty acids (FAs), i.e., ω-cyclohexyl or ω-cycloheptyl FAs. However, there are also species belonging to this genus that do not possess this trait. In addition, the difference of up to 9 % of the 16S rRNA gene sequences of 33 type strains compared with the first isolated type species, A. acidocaldarius DSM 446T, exceeds the 5.5 % threshold, which is typically used for the demarcation of a genus. This suggested that the taxonomy of the genus required a re-examination. We performed a phylogenomic and chemotaxonomic survey of the genus Alicyclobacillus. Phylogenetic trees based on the 16S rRNA gene and comparison of whole proteomes reconstructed from their genomes reveal that the genus Alicyclobacillus is reflected by two distinct clades; one represented by 14 type strains with a >94.1 % 16S rRNA gene similarity to A. acidocaldarius DSM 446T, and a second clade (composed of 14 type strains including A. cycloheptanicus) with a similarity of 91–94 %. This distinction fits well with their reported ability to produce alicyclic FAs, also revealed by the detection of two putative biosynthetic gene clusters (BGC) encoding their biosynthetic pathway in their genomes. The first BGC is responsible for the production of cyclohexane carboxylic acid-CoA and comprises five genes encoding proteins acting as an adjusted side branch of the shikimate pathway. The putative second BGC, responsible for production of cycloheptane carboxylic acid-CoA, comprises six genes encoding proteins acting as a side branch of the catabolic phenylacetic acid pathway. All Alicyclobacillus species of the first clade produce ω-cyclohexyl FAs and their genomes possess the corresponding BGC. In the second cluster, some species produce ω-cyclohexyl FAs, some produce ω-cycloheptyl FAs, whilst quite some members are not producing any alicyclic FAs. This corresponds to the presence or absence of the two BGCs in their genomes. We propose to reclassify all species of the second clade as members of a new genus, Paenalicyclobacillus gen. nov. and show that both Alicyclobacillus tengchongensis and Alicyclobacillus montanus are later heterotypic synonyms of Alicyclobacillus tolerans.