Aims: To describe three new Amycolatopsis strains and assess the antibiotic biosynthetic potential of the genus. Methods and Results: Three strains, designated S1.3(T), S3.6(T) and SE(8)3(T), belonging to the genus Amycolatopsis were isolated and found to cluster together by 16S rRNA and gyrB gene-based phylogenetic analysis. Genetic distance values, based on the gyrB gene, were calculated between the strains and their closest relatives and were all above the threshold value of 0.02 that has been proposed to distinguish Amycolatopsis type strains. DNA-DNA hybridization experiments against related type strains confirmed that strain S3.6(T) represents a unique genomic species. Strain S3.6(T) was also found to be distinct from strains S1.3(T) and SE(8)3(T), the latter two of which were also shown to be distinct from each other. Antibiotic biosynthetic genes were identified from multiple Amycolatopsis strains, and their presence was found to be phylogenetically associated. Conclusions: The data presented in this study indicate that strains S1.3(T), SE(8)3(T) and S3.6(T) belong to three novel species, for which the names Amycolatopsis circi sp. nov. (= DSM 45561(T) = NRRL B-24841(T)), Amycolatopsis equina sp. nov. (= DSM 45563(T) = NRRL B-24842(T)) and Amycolatopsis hippodromi sp. nov. (= DSM 45562(T) = NRRL B-24843(T)) are proposed. Significance and Impact of the Study: Three new species of Amycolatopsis are described, and the knowledge of the antibiotic biosynthetic potential of the genus has been extended.