AbstractUpon assembling the firstGossypium herbaceum(A₁) genome and substantially improving the existingGossypium arboreum(A₂) andGossypium hirsutum((AD)₁) genomes, we showed that all existing A-genomes may have originated from a common ancestor, referred to here as A₀, which was more phylogenetically related to A₁than A₂. Further, allotetraploid formation was shown to have preceded the speciation of A₁and A₂. Both A-genomes evolved independently, with no ancestor–progeny relationship. Gaussian probability density function analysis indicates that several long-terminal-repeat bursts that occurred from 5.7 million years ago to less than 0.61 million years ago contributed compellingly to A-genome size expansion, speciation and evolution. Abundant species-specific structural variations in genic regions changed the expression of many important genes, which may have led to fiber cell improvement in (AD)₁. Our findings resolve existing controversial concepts surrounding A-genome origins and provide valuable genomic resources for cotton genetic improvement.