Bulk sampling was conducted on the fiber （10 days after anthesis） of interspecific F₁ hybrids and parents of upland cotton to study the heterosis in fibers of interspecific F₁ hybrids in terms of phenotype and genetics and analyze the reasons for the unclear heterosis in traits of yield. Transcriptomics was used to analyze the significant differences in transcription levels between samples. Bioinformatics was used to identify the significant differences in metabolic pathways and key genes between samples. The results showed that the F₁ hybrid had fewer long fiber cells and the surface of the seed was covered by green fuzz. A total of 1 128 upregulated genes and 678 downregulated genes were identified in the transcriptome data of fiber between parents of upland cotton and interspecific F₁ hybrids. The results of analyzing gene enrichment showed that the differential expression genes （DEGs） were mainly enriched in the biosynthesis of phenylpropane and biosynthesis pathways of flavonoid. The results of parental allele-specific expression analysis of DEGs showed that the allele of fiber in interspecific F₁ hybrids tended to be expressed in sea- island cotton， among which the dihydroflavonol-3 '，5' - hydroxylase （F3'5'H） genes in the metabolic pathway of flavonoid in interspecific F₁ hybrids only expressed the alleles of sea-island cotton. Overall， the traits of fiber yield in the interspecific F₁ hybrids between sea-island cotton and upland cotton had negative heterosis and significant green and short fuzz in the fibers， which may be related to the secondary metabolites produced by the biosynthesis of phenylpropane and flavonoid. It is indicated that the heterosis and key metabolic pathways and genes of fibers in the interspecific F₁ hybrids between upland cotton and sea-island cotton are investigated at the transcription level. The relationship between phenotypic traits and transcription and the relationship between transcription and metabolic synthesis are established.