Abstract: 【Objective】 To explore the response of key genes to heat stress and compare the differences of gene expression. 【Methods】 RNA-Seq was used to sequence and analyze the transcriptome of YZ1 unde²r normal(26℃)and high temperature stress(42℃). 【Results】 Gohir.A08G10410A total of 383 up-regulated genes and 234 down regulated genes were identified in WN4/WH4, WN12/WH12, WN24/WH24 and WN48/WH48. According to GO function, the differential genes were mainly enriched into 20 categories, namely, up-regulated differential genes and down-regulated differential genes. According to KEGG function analysis, the differential genes were significantly enriched in flavonoid biosynthesis pathway, phenylalanine metabolism, circadian rhythm-plant and endoplasmic reticulum processing and other pathways. 39 transcription factors were identified, mainly belonging to WRKY and MYB families; Combined with gene annotation, it was found that the expression levels of these two genes were significantly different in the whole period. Gohir.A08G104100 was the main gene of heat stress, which was crucial to the heat tolerance of plants, while Gohir. D08G033300 was a limiting enzyme for plastid isoprene biosynthesis and was critical for chloroplast development. 【Conclusion】 Through comparative transcriptome analysis, the difference of gene transcription expression between upland cotton under high temperature stress and normal temperature is compared.