Highlights ► Genomes of three strains of mycobacteria producing 3,17-diketoandrostanes from phytosterol were sequenced. ► Genomes of AD and ADD producers are nearly identical, that of 9-OH-AD producer differs. ► Multiple kstD, kshA and kshB genes were found and annotated. ► AD and ADD-producing strains differ by a Lys135Ser substitution in Δ1 KSTD.