Abstract: 【Objective】 High-throughput gene chip analysis is performed using upland cotton with wide adaptability and high yield and sea island cotton with excellent fiber quality, aiming to screen and identify key genes involved in cotton fiber development. 【Methods】 The upland cotton SG747 (Gossypium hirsutum L.) and Egyptian cotton Giza75 (G. barbadense L.) were used as experimental materials. The Affymetrix Cotton Gene Chip was then used to perform a transcriptome analysis in developing fibers (10 Days after anthesis, DPA). 【Results】 The 3,905 transcripts obtained showed 2-fold or higher levels of expression changes between the two materials. Clustering analysis of these DE genes by Clusters of Orthologous Groups database was performed, which revealed that many genes were related to general function prediction (17.80%), translation, ribosomal structure and biogenesis (16.82%), posttranslational modification (14.32%). According to qRT-PCR analysis, it was speculated that Gra.2198.1.A1_at positively regulated cotton fiber development and Gra.85.1.S1_at, Ghi.249.1.A1_at, Ghi.8448.1.S1_x_at negatively regulated cotton fiber development, and the above genes might be involved in the development of cotton fiber. 【Conclusion】 We screened and identified four key candidate genes related to fiber development by using upland cotton and sea island cotton gene chips combined with qRT-PCR.