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Summary  Naturally coloured cotton (NCC) fibres need little or no dyeing process in textile industry to low‐carbon emission and are environment‐friendly. Proanthocyanidins (PAs) and their derivatives were considered as the main components causing fibre coloration and made NCCs very popular and healthy, but the monotonous fibre colours greatly limit the wide application of NCCs. Here a  G. hirsutum  empurpled mutant ( HS2 ) caused by T‐DNA insertion is found to enhance the anthocyanidins biosynthesis and accumulate anthocyanidins in the whole plant. HPLC and LC/MS‐ESI analysis confirmed the anthocyanidins methylation and peonidin, petunidin and malvidin formation are blocked. The deficiency of  GhOMT1  in  HS2  was associated with the activation of the anthocyanidin biosynthesis and the altered components of anthocyanidins. The transcripts of key genes in anthocyanidin biosynthesis pathway are significantly up‐regulated in  HS2 , while transcripts of the genes for transport and decoration were at similar levels as in WT. To investigate the potential mechanism of  GhOMT1  deficiency in cotton fibre coloration,  HS2  mutant was crossed with NCCs. Surprisingly, offsprings of  HS2  and NCCs enhanced PAs biosynthesis and increased PAs levels in their fibres from the accumulated anthocyanidins through up‐regulated  GhANR  and  GhLAR . As expected, multiple novel lines with improved fibre colours including orange red and navy blue were produced in their generations. Based on this work, a new strategy for breeding diversified NCCs was brought out by promoting PA biosynthesis. This work will help shed light on mechanisms of PA biosynthesis and bring out potential molecular breeding strategy to increase PA levels in NCCs.

Function deficiency of GhOMT1 causes anthocyanidins over‐accumulation and diversifies fibre colours in cotton ( Gossypium hirsutum )